The PI-3 kinase inhibitor, LY294002, blocks phosphorylation of Erk and MEK but not Ras–Raf-1 interaction. HPCs were harvested after stimulation with SF (50 ng/mL) for the indicated times (lanes 2 to 9). Prior to SF stimulation, serum-starved HPCs were incubated 30 minutes with 20 μM LY294002 (lanes 7 to 9). Lysates were subjected to affinity precipitation with GST-Raf-RBD prior to detection of activated Ras proteins by immunoblotting with anti-Ras antibodies (A). Cell lysates were subjected to SDS-PAGE and then immunoblotted with phospho-specific anti-MEK (B), phospho-specific anti-Erk and PKB (C), and anti-Erk2 antibodies (D).