Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Agonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assayAgonist activity at SMO in mouse NIH3T3 cells incubated for 2 hrs by Bright-Glo luciferase assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometerAntagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometer
Antagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometerAntagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometer
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometerAntagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometer
Antagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometerAntagonist activity at mouse SMO expressed in NIH/3T3 cells transfected with Gli-dependent luciferase-reporter after 15 hrs by luciferase reporter gene assay using luminometer
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of endogenous mouse Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of endogenous mouse Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Inhibition of endogenous mouse Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of endogenous mouse Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 1 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of wild type Smo (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of wild type Smo (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Inhibition of wild type Smo (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of wild type Smo (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH3T3 cells preincubated for 2 hrs followed by SAG stimulation and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449
Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449
Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449
Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449Antagonist activity at resistant Smo D473H mutant (unknown origin) in presence of GDC0449
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene methodAntagonist activity at Smo in mouse Shh-Light 2 cells assessed as inhibition of Shh-induced Gli1-reporter activity after 2 days by dual-luciferase reporter gene method
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Antagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assayAntagonist activity at smo in SAG-treated mouse Light2 cells after 48 hrs by renilla luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assayInhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo in mouse TM3 cells expressing Hh12 assessed as inhibition of 25 nM Ag1.5-induced Gli expression incubated for 30 mins prior to Ag1.5-induction measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Inhibition of endogenous human Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of endogenous human Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Inhibition of endogenous human Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of endogenous human Smo overexpressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Antagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assayAntagonist activity at human Smo receptor in HEPM cells assessed as inhibition of Gli expression after 24 hrs by quantigene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated for 1 to 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase reporter gene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expressionAntagonist activity at smoothened expressed in mouse Shh Light2 cells assessed as inhibition of purmorphamine- induced Gli-dependent luciferase gene expression
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Antagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathwayAntagonist activity at smoothened expressed in mouse Shh Light2 cells co-expressing Gli-dependent reporter gene assessed as inhibition of Hh pathway
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Inhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assayInhibition of Smo-mediated Hh signaling in human Shh-light2 cells by luciferase reporter gene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assayAntagonist activity at human SMO expressed in HEPM cells assessed as reduction of GLI expression after 24 hrs by Quantigene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at resistant Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity incubated for 24 hrs by dual luciferase assayAntagonist activity at resistant Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity incubated for 24 hrs by dual luciferase assay
Antagonist activity at resistant Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity incubated for 24 hrs by dual luciferase assayAntagonist activity at resistant Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity incubated for 24 hrs by dual luciferase assay
Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.
Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.
Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.Assessing differentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblastsby measuring AP enzymatic activity as a readout for Smo agonist activity.
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.
Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.
Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.Measuring inhibition of Hh pathway signalling in Gli-dependent reporter assays in Shh-LIGHT2 cells.
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Agonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayAgonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Agonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayAgonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Agonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayAgonist activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 500 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 500 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 500 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 500 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from mouse SMO transfected in human HeLa cells by competition binding assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 50 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 50 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 50 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 50 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Displacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assayDisplacement of BODIPY-labeled cyclopamine from human SMO transfected in human HeLa cells by competition binding assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Agonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factorAgonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factor
Agonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factorAgonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factor
Agonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factorAgonist activity at Smo in mouse NIH/3T3 cells transfected with Gli-dependent firefly luciferase reporter assessed as stimulation of Gli transcription factor
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 100 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 100 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 100 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)Inhibition of Smo in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh pathway activity by measuring SAG-EC50 at 100 nM measured after 36 hrs by Gli-luciferase reporter gene assay (Rvb = 2.93 nM)
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrsInhibition of SMO-mediated Hedgehog signaling pathway in mouse C3H10T1/2 cells assessed as reduction in sonic hedgehog-induced osteoblast differentiation by measuring alkaline phosphatase activity incubated for 72 hrs
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Inhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysisInhibition of Smo expressed in mouse C3H10T1/2 cells assessed as reduction in alkaline phosphatase level after 72 hrs by spectrophotometric analysis
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Activity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assayActivity at Smo in mouse C3H10T1/2 cells assessed as induction of cell differentiation into osteoblast incubated for 6 days by alkaline phosphatase assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Antagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assayAntagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assay
Antagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assayAntagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamineInhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamine
Inhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamineInhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamine
Inhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamineInhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamine
Inhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamineInhibition of human recombinant SMO expressed in mouse C3H10T1/2 cells assessed as inhibition of association of BODIPY-cyclopamine
Antagonist activity at mouse Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assayAntagonist activity at mouse Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assay
Antagonist activity at mouse Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assayAntagonist activity at mouse Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assayInhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assay
Inhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assayInhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from human Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assayDisplacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assayInhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assayInhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assay
Inhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assayInhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopyInhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopy
Inhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopyInhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopy
Inhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopyInhibition of BODIPY-cyclopamine binding to Smo expressed in HEK293T cells after 1 hr by fluorescence microscopy
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assayInhibition of SMO-mediated hedgehog signalling pathway in human HPEM cells by luciferase reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assayInhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assay
Inhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assayInhibition of Bodipy-cyclopamine binding to mouse FLAG-tagged Smo expressed in HEK293 cells after 3 hrs by Hoechst staining based fluorescence assay
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assayAntagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assay
Antagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assayAntagonist activity at mouse cloned Smo receptor expressed in NIH-3T3 cells co expressing Gli1 binding site after 15 hrs by luciferase reporter gene assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at human Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assayAntagonist activity at human Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assay
Antagonist activity at human Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assayAntagonist activity at human Smo expressed in CHO cells assessed as inhibition of BODIPY-cyclopamine binding after 4 hrs by fluorescence assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysisInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysisInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysisInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysisInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced Gli1 transcriptional activity after 48 hrs by RT-PCR analysis
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assayInhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assay
Inhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assayInhibition of human Smo expressed in human U2OS cells assessed as reduction in BODIPY-cyclopamine fluorescence signaling by competitive displacement assay
Binding affinity to human SMO (181 to 787 residues) expressed in human HEK293 cells assessed as radioactivity incubated for 2 hrs in presence of H3-labeled smo antagonist by ligand-displacement assayBinding affinity to human SMO (181 to 787 residues) expressed in human HEK293 cells assessed as radioactivity incubated for 2 hrs in presence of H3-labeled smo antagonist by ligand-displacement assay
Binding affinity to human SMO (181 to 787 residues) expressed in human HEK293 cells assessed as radioactivity incubated for 2 hrs in presence of H3-labeled smo antagonist by ligand-displacement assayBinding affinity to human SMO (181 to 787 residues) expressed in human HEK293 cells assessed as radioactivity incubated for 2 hrs in presence of H3-labeled smo antagonist by ligand-displacement assay
Antagonist activity at SMO D473H mutant (unknown origin) expressed in mouse TM3 cells coexpressing Gli-luc reporter gene assessed as inhibition of Hh-Ag1.5 induced Gli luciferase activity incubated for 48 hrs by luminescence assayAntagonist activity at SMO D473H mutant (unknown origin) expressed in mouse TM3 cells coexpressing Gli-luc reporter gene assessed as inhibition of Hh-Ag1.5 induced Gli luciferase activity incubated for 48 hrs by luminescence assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Displacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assayDisplacement of boron-dipyrromethene-cyclopamine from human smoothened receptor expressed in HEK293 cells incubated for 4 hrs by hSMO-BC binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Inhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assayInhibition of SMO in mouse 3T3/HH FlashII-7 cells incubated for 36 hrs in presence of SHH by Gli-luciferase reporter assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopyDisplacement of BODIPY-labelled cyclopamine from human Smo receptor expressed in HEK293 cells after 2 hrs by fluorescence microscopy
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Inhibition of Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assayInhibition of Smo D473H mutant (unknown origin) expressed in mouse C3H 10T1/2 cells cotransfected with Gli reporter gene assessed as Gli luciferase activity by dual luciferase assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic methodDisplacement of BODIPY-Cyclopamine from human HA-tagged Smo receptor expressed in human U2OS cells at 1 to 10000 nM incubated for 2 hrs by DAPI staining based fluorescence microscopic method
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assayInhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assay
Inhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assayInhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assay
Inhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assayInhibition of Smo D473H mutant (unknown origin) expressed in mouse NIH3T3 cells cotransfected with Gli luciferase reporter gene assessed as Gli luciferase activity incubated for 48 hrs by Bright-Glo reagent based luciferase assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at human Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assayInhibition of Smo in mouse C3H10T1/2 cells assessed as inhibition of human Shh-induced cell differentiation after 48 hrs by alkaline phosphatase reporter assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Antagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assayAntagonist activity at Smo receptor in mouse NIH/3T3 cells harboring GRE-Luc assessed as inhibition of SAG-induced Hh signaling pathway preincubated with cells followed by SAG addition measured after 48 hrs by luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assayInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli-responsive betagalactosidase activity after 30 hrs by BetaGLo assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Inhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assayInhibition of Smoothened transfected in HEK293 cells by transient transfection cell-based assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assayAntagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assay
Antagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assayAntagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysisDisplacement of Bodipy-labelled cyclopamine from Smo expressed in COS-1 cells in presence of 2% FBS after 4 to 6 hrs by FACS flow cytometric analysis
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Inhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysisInhibition of of Bodipy-labelled cyclopamine binding to Smo expressed in COS-1 cells in presence of 20% NHS after 4 to 6 hrs by FACS flow cytometric analysis
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Inhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assayInhibition of Smo in mouse C3H10T1/2 cells using human recombinant SHH assessed as effect on SMO/SHH transient transcriptional activation after 20 hrs by Gli-luciferase reporter assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activityInhibition of Smo in mouse Ptch-deficient MEF cells assessed as inhibition of Shh-induced Gli1 transcriptional activity
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 20% normal human serum
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assayInhibition of Smo expressed in mouse NIH/3T3 cells after 20 hrs by Gli reporter gene assay
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Antagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assayAntagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assay
Antagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assayAntagonist activity at mouse FLAG-tagged Smo D477G mutant expressed in mouse Shh Light2 cells assessed as inhibition of SAG-induced Hh signaling pathway after 48 hrs by dual luciferase reporter gene assay
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 2% fetal calf serum
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 1 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Antagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assayAntagonist activity at SMO in mouse NIH/3T3 cells harboring Gli-responsive firefly luciferase gene assessed as inhibition of SAG-induced response preincubated for 2 hrs followed by SAG addition and measured after 24 hrs by Bright-Glo luciferase assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assayDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells incubated for 3 hrs by fluorescence competitive displacement assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Inhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR methodInhibition of smo-mediated hedgehog signaling pathway in mouse ASZ001 cells assessed as decrease in Gli1 mRNA expression after 48 hrs by qPCR method
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Antagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assayAntagonist activity at mouse Smo receptor expressed in CHO cells by [3H]Hh-Ag binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Inhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assayInhibition of SMO D473H mutant receptor (unknown origin) assessed as inhibition of SAG-induced Hh signaling pathway by Gli luciferase reporter cell-based assay
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Displacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiographyDisplacement of [125I]-PA cyclopamine from smoothened expressed in african green monkey COS-1 cells after 10 mins by autoradiography
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Inhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assayInhibition of SMO in mouse NIH/3T3 cells assessed as inhibition of SAG-induced hedgehog-mediated luminescence signaling by GRE-luciferase reporter gene assay
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serumDisplacement of BODIPY-cyclopamine from human Smo expressed in HEK293 cells in presence of 2% fetal calf serum
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from human Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Inhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCSInhibition of BODIPY-cyclopamine to Smo in human HEK293 Flag-Smo cells in presence of 2% FCS
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Displacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assayDisplacement of [3H]3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide from mouse Smoothened receptor expressed in CHO-K1 cells by membrane filter binding assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Inhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assayInhibition of Smo receptor (unknown origin) expressed in NIH3T3 cells assessed as inhibition of Smo agonist SAG-induced GRE activation after 30 hrs by luciferase reporter gene assay
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).Radioligand Competition Binding Assay: For the binding competition assay, 100 μL of Assay Buffer was added to all the wells of a 96 well GF/B filter plate (Millipore MultiScreen-HTS-FB cat# MSFBN6B50) for 10 minutes to pre-wet the filter prior to evacuation of the buffer (8 inches Hg for 8 seconds). To the pre-wet wells is added: 20 μL of Assay Buffer, 10 μL diluted test agent, 20 μL of a tritiated Smo antagonist (15 nM stock solution), and 50 μL of membrane preparation (40 μg total protein per well). The plates are sealed and mixed at room temperature for 5 min, incubated at room temperature for 2 hours, then washed 5 times with 100 μL/each of wash buffer and vacuum dried for 8 seconds at 8 inches Hg. The plate is then dried for one hour in a 60° C. oven prior to the addition of 45 μL of Microscint 20 (Packard, #6013621) to each well and incubation at RT for 30 minutes to 1 hour. The plate is counted in a TopCount scintillation counter (Perkin Elmer).
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Inhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assayInhibition of Smoothened receptor in mouse TM3 cells transfected with pTA-8xGli-Luc reporter gene assessed as inhibition of 25 nM 3-chloro-4,7-difluoro-N-(4-methoxy-3-(pyridin-4-yl)benzyl)-N-(4-(methylamino)cyclohexyl)benzo[b]thiophene-2-carboxamide-induced Hedgehog pathway activation by Gli shift assay
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Displacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serumDisplacement of BODIPY-cyclopamine from Smo expressed in COS-1 cells after 4 to 6 hrs by Flow Cytometry analysis in presence of 20% normal human serum
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to Smo D473H mutant expressed in U2OS cells by scintillation countingBinding affinity to Smo D473H mutant expressed in U2OS cells by scintillation counting
Binding affinity to Smo D473H mutant expressed in U2OS cells by scintillation countingBinding affinity to Smo D473H mutant expressed in U2OS cells by scintillation counting
Binding affinity to Smo D473H mutant expressed in U2OS cells by scintillation countingBinding affinity to Smo D473H mutant expressed in U2OS cells by scintillation counting
Binding affinity to wild type Smo expressed in U2OS cells by scintillation countingBinding affinity to wild type Smo expressed in U2OS cells by scintillation counting
Binding affinity to wild type Smo expressed in U2OS cells by scintillation countingBinding affinity to wild type Smo expressed in U2OS cells by scintillation counting
Binding affinity to wild type Smo expressed in U2OS cells by scintillation countingBinding affinity to wild type Smo expressed in U2OS cells by scintillation counting
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Binding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysisBinding affinity to recombinant human Smo receptor assessed as dissociation constant at equilibrium by SPR analysis
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometryDisplacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometry
Displacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometryDisplacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometry
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from Smo D473H mutant expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysisDisplacement of BODIPY-LY from SMO (unknown origin) expressed in HEK293F cells measured after 15 mins by flow cytometric analysis
Displacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometryDisplacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometry
Displacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometryDisplacement of [3H]-cyclopamine from human wild-type SMO receptor expressed in HEK293T cell membranes by liquid scintillation spectrometry
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation countingDisplacement of [3H]-cyclopamine from SMO V404M mutant in gefitinib resistant human HCC827 cells by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting
Displacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation countingDisplacement of [3H]cyclopamine from wild type Smo expressed in U2OS cells after 2 hrs by scintillation counting