Figure 8. Time-dependent effects of imatinib on PDGF-induced versus insulin-induced Akt/GSK-3β/p70S6kinase phosphorylation. Serum-deprived (24 h) RGC-5 cells were pretreated without (control) or with 10 µM imatinib for increasing time intervals (3 h and 24 h). Subsequently, control and imatinib-treated cells were stimulated with either 30 ng/ml PDGF or 30 nM insulin for 6 min. The cell lysates were subjected to immunoblot analysis for Akt, GSK-3β, and p70S6kinase phosphorylation (A-C, and D-F) using the indicated primary antibodies (see Methods). To normalize the changes in protein phosphorylation in the immunoblots, we used β-actin as an internal control. Note: For data analyses, PDGF- or insulin-induced protein kinase phosphorylation in the absence of imatinib was normalized to 100%. The respective bar graphs shown are the mean±SEM values from 3 to 4 experiments. The asterisk indicates a p<0.05 compared with the respective PDGF-induced protein kinase phosphorylation.