Figure 1

Analysis of Endogenous Ras-dependent Raf Dimerization and Activation (A) Serum-starved HeLa cells were treated or not with EGF for five minutes prior to lysis. Endogenous A-Raf, B-Raf, or C-Raf proteins were immunoprecipitated and probed for the binding of other endogenous Raf members. (B) Serum-starved HeLa cells were treated or not with EGF prior to fixation. Red foci indicate binding interactions in the PLA assay. Cell nuclei were stained with DAPI. (C) HeLa cells stably expressing the pLKO.1 shA-Raf, shB-Raf, shC-Raf or empty vector were serum-starved and then treated or not with EGF prior to lysis. Endogenous Raf proteins were immunopurified and their intrinsic kinase activity determined using kinase-inactive MEK as a substrate. Error bars represent the standard deviation of the mean for three independent experiments.