Figure 7.

W-13 inhibits Raf activity. COS-1 cells serum starved for 24 h were incubated with W12 or W-13 (10 μg/ml) for 60 min and/or 25 ng/ml EGF for the last 10 min at 37°C. Raf activity was measured, as described in MATERIALS AND METHODS, from the lysates by immunoprecipitation with anti-Raf antibody and kinase cascade assay with GST-MEK, GST-ERK2, and MBP as substrates. (A) A representative experiment assayed in triplicate and error bars represent SD (n = 3). (B) By coimmunoprecipation, there is no effect in the Ras-Raf interaction after W-13 treatment. In C, lysates from the same experiment were probed with anti–P-MEK.