FIG. 4.

Effect of PI3-kinase on Ras activity. COS (A) or NIH 3T3 (B) cells were serum starved and either preincubated with dimethyl sulfoxide (cont), 20 μM LY294002 (LY), or 100 nM wortmannin (Wort) for 20 min and then stimulated with EGF (10 ng/ml) (COS cells) or PDGF (50 ng/ml) (NIH 3T3 cells) for the indicated times. Active GTP-bound Ras (upper rows) was extracted from the lysates and detected as described in Materials and Methods. An immunoblot of 5% total Ras in the extracts is shown as a loading control (lower rows). Blots are for one representative experiment performed three times with similar results.