A luciferase assay was performed to compare the effect of PTCH1, PTCH2 and PTCHD1 on Gli-dependent transcription with a previously described method (39). Briefly, the 10T1/2 cells were transiently transfected with mixtures containing 0.1 μg β-galactosidase to normalize for transfection efficiency, 1 μg reporter plasmid (8xGlipro) encoding multimerized Gli binding sites fused to the luciferase gene and up to 1 μg of Gli2, PTCH1 or PTCH2 or PTCHD1. Gli-dependent transcription was measured and normalized by β-galactosidase. Data were replicated in independent experiments performed in triplicates. In another assay, 10T1/2 cells were transiently transfected with mixtures containing 0.1 μg β-galactosidase, 1 μg 8xGlipro reporter plasmid and purmorphamine, PTCH1 or PTCH2 or PTCHD1. The effect of PTCH1, PTCH2 and PTCHD1 on the endogenous Gli-dependent transcription was measured. Statistical significance was calculated as p below 0.05, using the Student’s t-test.
