Fig. 2. SHH signaling stabilizes IRS1 protein. (A) RNA was collected from SHH-treated CGNPs and RT-PCR analysis was conducted for IRS1, cyclin D2 and β-actin to verify equal RNA input. (B) qPCR was used to quantify levels of IRS1 expression. Expression levels of IRS1, depicted as arbitrary units, do not change in response to SHH treatment. (C) CGNPs were treated with SHH, cyclopamine or lactacystin (10 µM) for indicated times. Levels of IRS1 protein are stabilized in the presence of the lactacystin and cyclopamine. (D) Western blot analysis of IRS1 protein levels in CGNPs infected with GLI1 retroviruses and subsequently cultured without SHH for 36 hours. GLI1 infection can sustain proliferation as indicated by cyclin D2 levels and as previously reported (Oliver et al., 2003). IRS1 was present but at lower levels than in CGNPs infected with GFP-expressing retroviruses and treated with exogenous SHH, suggesting the existence of GLI-mediated and non-GLI-mediated mechanisms that promote IRS1 protein accumulation.