Figure 1. Effect of dimer interface R > H mutation and Raf inhibitor treatment on Raf dimerization. (A and B) Serum-starved HeLa cells expressing Flag-tagged WT- R > H-, E > K, R > H/E > K-C-Raf proteins were treated or not with EGF for 5 min prior to lysis. Immunoprecipitated Flag-C-Raf was probed for the binding of endogenous B-Raf (A and B) and for the phosphorylation of S338 (A). The kinase activity of purified Flag-C-Raf proteins was determined in immune complex kinase assays using recombinant MEK as a substrate (A). ³²P-labeled MEK was analyzed by SDS-PAGE and autoradiography. (C) Cycling HeLa cells expressing the indicated Flag or Pyo-tagged Raf proteins were treated with PLX4720 or SB590885 for 1 h prior to lysis. Immunoprecipitated Flag-Raf proteins were probed for the binding of Pyo-Raf.