SCF and inhibitors of MEK and PI-3 kinase have no effects on EPO-induced activation of STAT5. (A) Serum-starved day-8 ECFC were treated with SCF, EPO, or SCF + EPO for 5, 10, and 30 minutes. Nuclear extracts were extracted and analyzed for STAT activity with a³²P-labeled PIE oligonucleotide probe. EPO* (lane 12, A) denotes a supershift analysis with the nuclear extracts preincubated with anti-STAT5b antibody (0.2 μg) for 10 minutes at room temperature before the addition of the PIE probe. (B) Serum-starved cells were pretreated with 100 μmol/L PD98059 or 0.5 μmol/L wortmannin for 1 hour before stimulation with EPO for 10 and 60 minutes. STAT5 activity was determined as described above.