Peptide Competition and Mutant Analysis:
Immunoprecipitates prepared from Hek293 cells stimulated with EGF and overexpressing wild-type c-Raf (1-4) or c-Raf mutant S621A (5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4^oC, then were incubated with 0.50 µg/mL ab4767 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: the phosphopeptide immunogen (1), a generic phosphoserine containing peptide (2), the non-phosphopeptide corresponding to the immunogen (3), or, no peptide (4, 5). The smaller blot presented below shows the relative amount of protein in the wild-type vs. the S621A mutant protein via a c-Raf pan antibody. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that only the peptide c