Fig. 2.

ERAP1 activates Hh signaling by impairing βTrCP protein expression. a–f Representative immunoblotting analyses of the indicated proteins in MEFs transfected with increasing amounts of vector encoding ERAP1 (a, d) or treated for 24 h with Leu-SH at the indicated concentration (b, e), or SAG (200 nM) and Leu-SH (30 μM) (c), or DTT as control. In f MEFs were transduced with shCTRL or shERAP1 and transfected with a vector encoding ERAP1. Actin (a–c, f) and tubulin (d, e) were used as loading controls. g ERAP1, Gli1 and βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 in the presence of small interfering RNAs (siRNAs) to a non-relevant mRNA (siCTRL) or murine βTrCP mRNA (siβTrCP). h βTrCP protein levels in MEFs transfected with an empty vector or a vector encoding ERAP1 and treated with cycloheximide (CHX, 100 µg/mL) at different time points. Densitometry analysis of actin-normalized βTrCP values of three independent experiments is shown (right panel). i, j Endogenous βTrCP was immunoprecipitated from MEFs expressing the indicated proteins and treated with MG132 (50 μM) for 4 h (i) or increasing doses of Leu-SH for 24 h (j), followed by immunoblotting with an anti-HA antibody to detect conjugated HA-Ub. Blots were both reprobed with a βTrCP antibody. Bottom ERAP1 and βTrCP protein levels in total cell lysate. Actin was used as loading control. k Immunoblotting (upper panel) and densitometric analysis (lower panel) of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs and treated after 24 h with increasing amount of Leu-SH for 24 h. l Immunoblotting analysis of HA-Gli1 WT or HA-Gli1ΔC protein levels transfected in MEFs transduced with shCTRL or shERAP1. Actin was used as loading control