FIG. 2.

SFK activity is required for full activation of EGF-dependent MAPK activity. GST-Erk-2-transfected Cos1 cells were serum starved (serum free [SF]) for 16 h prior to being treated with SFK inhibitor PP1 (10 μM) or vehicle (DMSO) for 2 h. Cells were either left unstimulated (lane 0) or stimulated (Stim.) with EGF at 100 ng/ml for indicated times (2, 5, or 10 min). GST-Erk-2 was purified from cleared lysates with glutathione Sepharose, and this exogenous Erk-2 was assayed for activation via immunoblotting with phospho-specific MAPK antibody. Inhibition of SFKs (PP1) but not vehicle dramatically decreases EGF-dependent MAPK activation in Cos1 cells. Differences in MAPK activation could not be attributed to total Erk-2 levels (anti-pan-MAPK). Treatment with PP1 at 10 μM does not influence activation of the EGFR (anti-pY EGFR).