FIG. 2.

The PI 3-kinase inhibitor LY294002 blocks Ras activation induced by low concentrations of EGF. (A) Serum-starved COS-7 cells were either left untreated or preincubated with LY294002 (LY; 20 μM) or wortmannin (WM; 100 nM) for 5 min and then stimulated with increasing concentrations of EGF for 5 min. Active GTP-bound Ras was extracted from lysates with the GST-Raf-Ras-binding domain (RBD) coupled to glutathione agarose and analyzed by immunoblotting with pan-Ras antibodies. (B) The effect of 20 μM LY294002 on endogenous Ras GTP-loading and endogenous ERK2 activation expressed as percent inhibition of activation seen at different concentrations of EGF. (C) Ras activation is not affected by matrix composition. COS-7 cells were attached to uncoated tissue culture dishes (−) or to dishes that had been precoated with either fibronectin (FN) or collagen (CO). Serum-starved cells were then either left untreated or preincubated for 5 min with LY294002 (LY; 20 μM) before being challenged with 0.05 ng of EGF per ml for 5 min. The amount of GTP-bound Ras in lysates was determined as described for panel A. Representative experiments are shown.