Fig. 1. IRS1 protein is upregulated in proliferating CGNPs. (A) CGNP cultures were prepared from different litters on different dates. Preparations of the cells were treated with SHH or left untreated for 24 hours prior to lysis. The autoradiograph depicts a western blot for several downstream components of IGF signaling pathway and the cell cycle progression marker cyclin D2. Only IRS1 levels are upregulated in SHH-treated samples, which correlates with increases in cyclin D2. β-Tubulin demonstrates equal protein loading. (B) The autoradiographs show western blots for CGNPs treated with two SHH signaling pathway inhibitors, forskolin (10 µM) or cyclopamine (1 µg/ml), for increasing time points. In the absence of continuous SHH signaling, levels of IRS1 decrease, which also correlates with decreased cyclin D2 expression. (C) SHH-treated CGNPs were fixed and immunostained for IRS1 (red) and p27 (green). IRS1 and p27 mark different populations of cells. Confocal imaging (right panel) confirms IRS1 presence in the nucleus (arrowhead) and cytoplasm (arrow). (D) Left: cerebellar section from post-natal day 7 mouse immunostained for IRS1 (red) and GFAP (green). Middle (low power) and right (high power): SHH-treated primary CGNP cultures immunostained for IRS1 (red) and GFAP (green). IRS1 is excluded from glia both in vivo and in vitro. (E) In the left (low power) and middle (high power) panels, PN 7 mice were pulsed with BrdU and stained for BrdU (green) and IRS1 (red). IRS1 colocalizes to proliferating CGNPs in the EGL. In the far right panel, IRS1 (red) is expressed in the cytoplasm of BrdU-positive cells (green) in CGNP cultures.