Figure 3

Silencing of InsR enhanced PIK3/Akt signaling while suppressing Ras/Erk1/2 activities in MES-13 mesangial cells. (A) Cell lysates were immunoprecipitated with an antibody againts phosphorylated-tyrosine, and PI3K activity was determined with in vitro lipid kinase assay. PIP, Phosphoinositide 3-phosphate, the phosphorylated end product. Ori, origin of migration in thin-layer chromatography (upper panel). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments (lower panel). (B) Cell lysates were subject to Western blotting with antibodies against phosphorylated-Akt (P-Akt, Thr308), total-Akt (T-Akt), phosphorylated-p70S6K (P-p70S6K) and total-p70S6K (T-p70S6K). The bar graphs show the densitometric scanning results (means ± SEM) from four independent experiments. (C) Lysates were immunoprecipitated with an immobilized anti-phospho-Akt (Ser473) antibody, and the kinase reaction was carried out in the presence of cold ATP and GSK-3α/β fusion protein as described. An image of gel stained after transfer was shown as a loading monitor (middle panel). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. (D) Ras activation was evaluated by pulling down active GTP-loaded Ras with a GST fusion protein containing the Ras binding domain of Raf-1 (GST-Raf1-RBD) followed by blotting with an anti-Ras antibody (upper panel). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. (E) Cell lysates were subject to Western blotting with antibodies against phosphorylated-Erk1/2 (P-Erk1/2) and total Erk1/2 (T-Erk1/2). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. *, P<0.05 vs. SC transfected cells.