PI-3 kinase activity is required for activation of Raf. Effect of SF (50 ng/mL) stimulation on Raf-1 phosphorylation (lanes 2 and 3). HPCs were pretreated with 20 μM LY294002 (lane 3) for 30 minutes. (A) Lysates were precipitated with GST-Raf-RBD and immunoblotted with anti-Ras antibody. (B) Control immunoblot analysis with anti-Ras. (C) Lysates were immunoprecipitated with anti–Raf-1 antibodies and immunoblotted with anti–Raf-1 antibodies. (D) Control immunoblot analysis of lysates with anti-Erk2. (E) Antiphospho-MEK analysis of the lysates. (F) Antiphospho-specific (S259) Raf-1 immunoblot analysis of lysates. (G) Anti-Ras immunoblot analysis of lysates. (H) In vitro Raf kinase activity. Values (SEM) are calculated from 3 independent experiments performed as described in “Materials and methods.”