Evc2 is a positive regulator of Hh signalling. (a and b). Representative reporter assays in LIGHT2 Gli-reporter cells (a) and MC3T3 cells (b) transfected with siRNA and treated with control DMSO (white bars) or Hh agonist purmorphamine (black bars). Purmorphamine increases expression of the Gli reporter (FF Luc) with respect to the Renilla control (Ren Luc). Evc2 knockdown was confirmed by western blotting (lower images). Evc2 (approximately 140 kDa) was detected using R1656 and α tubulin (50 kDa) was used as a loading control. Cells with reduced Evc2 protein levels (approximately 80% less than normal) have a significant reduction in Gli reporter expression in response to purmorphamine compared to those transfected with the non-targeting siRNA pool. (c). The purmorphamine induced expression of the Hh responsive gene, Ptch1, is significantly diminished in Evc2 null MEFs (-/-) compared to wild-type controls (+/+) as assessed by RT-PCR.