MEK inhibitor PD98059 and PI-3 kinase inhibitor wortmannin inhibit synergistic activation of MAPK (A) and cell growth (B and C). Serum-starved cells were left untreated (control) or pretreated with 100 μmol/L PD98059 or 0.5 μmol/L wortmannin for 1 hour and then stimulated with SCF and EPO separately or jointly for 10 minutes. Whole cell extracts were subject to SDS-PAGE and Western blotting with phospho-specific MAPK antibody or anti-MAPK antibody. The latter detects both phosphorylated and nonphosphorylated MAPK. A representative figure from four experiments with similar results is shown. Each experiment was performed with blood samples from different normal volunteers. It should be noted that a nonspecific band right below phospho-Erk2 was equally distributed in all lanes. To analyze the effects of the inhibitors on erythroid cell growth, day-6 ECFC (5 × 10³) were allowed to grow for another 7 days in liquid medium supplemented with EPO (○) or SCF + EPO (•) in the presence of varying concentrations of PD98059 (B) or wortmannin (C). Cell numbers were determined after 7 days in culture. Results represent data from three independent experiments.