Inhibition of Raf-1 hyperphosphorylation by PD98059 increases Raf-1 kinase activity. (A) Raf-1 kinase assay. BaF3-EpoR cells were serum-starved overnight and were incubated in the absence or presence of inhibitor PD98059 for 10 minutes, then in the absence or presence of EPO for 5 minutes. Cell lysates were immunoprecipitated with anti–Raf-1 antibody, and the immunoprecipitate was used for Raf-1 kinase assay. Four microliters of the first-stage reaction of kinase assay was used to phosphorylate MBP. Black bars show increase above background. Error bars represent means ± SD. (B) Western blot analysis of the first-stage reaction from kinase assay.