Fig. 2. Tryptic phosphopeptide mapping of B-Raf. (A) Metabolic labeling and immunoprecipitation of B-Raf and B-RafAA. COS1 cells grown on a 60 mm plate were transfected with wild-type B-Raf (lane 1), B-Raf + HRasV12 (lane 2) or B-RafAA + HRasV12 (lane 3), labeled with [32P]orthophosphate, and immunoprecipitated with anti-HA antibody. Immunocomplexes were separated by SDS–PAGE and visualized by antoradiography. Positions of size markers are indicated in kilodaltons (KD) on the right. An immunoblot of the above immunoprecipitation using anti-HA antibody is shown at the bottom. (B, C and D) Phosphopeptide mappings for wild-type B-Raf, B-Raf + HRasV12 and B-RafAA + HRasV12, respectively. The 32P-labeled bands were excised from the membrane, digested with trypsin and analyzed by two-dimensional thin-layer electrophoresis. The directions for electrophoresis (E, from cathode to anode) and TLC (T) are indicated in the lower left corner of each panel. Arrows point to positions of phosphopeptides for references between different panels. Dashed circles indicate phosphopeptides present in (C) that are missing in (D). Open arrowheads indicate the Ras-induced phosphopeptide not affected in B-RafAA. (E) Phosphorylation of Thr598 and Ser601 determined by specific anti-phospho Thr598 and Ser601 antibodies. COS cells were transfected with pcDNA (vector, lane 1), wild-type B-Raf (lane 2), co-transfected with wild-type B-Raf and HRasV12 (lane3), B-RafAA (lane 4) or co-transfected B-RafAA and HRasV12 (lane 5). Immunoprecipitates of B-Raf and B-RafAA were isolated and resolved by SDS–PAGE, transferred to membrane, and blotted with anti-phospho Thr598 (top panel) or anti-phospho Ser601 antibody (middle panel). Protein loading was examined by blotting using HA antibody (bottom panel).