Fig. 1

ERAP1 positively regulates the Hh pathway at postreceptor level. a Luciferase activity of NIH3T3 Shh-Light II cells treated for 24 h with SAG and increasing amounts of Leu-SH or DTT as control. b, c Quantitative real-time PCR (qRT-PCR) (b) and representative immunoblotting (c) analyses of Gli1 expression in the NIH3T3 murine fibroblasts transduced with lentiviral vectors encoding either control shRNA (shCTRL) or ERAP1 shRNA (shERAP1#1 and shERAP1#2) and treated with SAG or DMSO for either 24 or 48 h. In c ERAP1 expression was also evaluated and actin was used as loading control. d, f qRT-PCR analysis of Hh target genes expression in Ptch^−/− (d) and SuFu^−/− MEFs (f) both treated with Leu-SH (30 μM) or DTT as control. e Representative model of the constitutive activation of Smo or Gli1 in Ptch^−/− and SuFu^−/− MEFs, respectively. g, h qRT-PCR analysis of Hh target genes expression in Ptch^−/− (g) and SuFu^−/− MEFs (h) transduced with shCTRL or shERAP1 constructs. Data in b, d, f, g, and h are normalized to endogenous GAPDH and HPRT controls and expressed as the fold change respect to the control sample value. All data represent the mean of three independent experiments. Mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001 calculated with two-sided Student’s t-test