Fig. 7. Effects of inhibitors on IGF-I-induced activation of the PI 3-kinase and MAP kinase signaling pathways. (a) Serum-starved cells (0). Cells not exposed to inhibitors (IGF-I) and pre-treated with 10 µM LY 294002 for 30 minutes (LY+IGF-I) or 40 µM PI analog for 3 hours (PI analog+IGF-I) were stimulated with 50 ng/ml IGF-I for the indicated times. Phosphorylation of PKB/Akt was detected with an antibody against phospho-Akt. For a loading control, the same blots were stripped and re-probed with an antibody against Akt (Materials and Methods). One of the re-probed blots is shown. (b). Serum-starved cells (0). Cells not exposed to inhibitors (IGF-I) and pre-treated with 50 µM PD 98059 (PD+IGF-I) or 50 µM UO 126 (UO+IGF-I) for 30 minutes were stimulated with 50 ng/ml IGF-I for the indicated times. The ERK1/2 MAPK phosphorylation was detected with specific phospho-MAPK antibodies. The same blot was stripped and re-probed with antibody against total ERK1/ERK2 (p44/p42) (Materials and Methods).