Dominant-negative Ras N17Ras does not inhibit the EPO-dependent induction of Raf-1 activity. (A) Raf-1 kinase assay. BaF3-EpoR cells were cotransfected with 10 μg Raf-1 and 30 μg dominant-negative Ras (N17 Ras) expression plasmid DNA. Cells were incubated in the absence or presence of EPO for 5 minutes. Cells were lysed, and Raf-1 was immunoprecipitated with antiflag antibody. The immunoprecipitate was used for the Raf-1 kinase assay. Four microliters of the first-stage reaction of the kinase assay was used to phosphorylate MBP. Error bars represent means ± SD. (B) Western blot analysis of the first-stage reaction from kinase assay.