Synergistic effects of SCF and EPO on phosphorylation of MAPK (ERK1 and ERK2) (A) and MEK (B) and on stimulation of MAPK activity (C). Serum-starved ECFC were treated with SCF, EPO, or SCF + EPO for the indicated periods of time. Whole cell extracts (20 μg protein) were subject to SDS-PAGE and Western blot analyses with anti–phospho-MAPK (A) or anti–phospho-MEK (B) antibodies. (C) For determination of MAPK activity, cells were stimulated for 10 minutes with SCF, EPO, or SCF + EPO and whole cell extracts containing about 60 μg of total protein were subjected to immunoprecipitation with anti–phospho-MAPK. The MAPK activities in the immunoprecipitates were analyzed by using myelin basic protein as a substrate in the presence of [γ-³²P]-ATP in buffer A containing PKI peptide and calmidizolium, inhibitors of PKA and PKC, respectively. A representative figure from four experiments with similar results is shown. Each experiment was performed with blood samples from different normal volunteers.