EPO-induced hyperphosphorylation of Raf-1 requires active MEK. (A) BaF3-EpoR cells were treated with specified concentrations of PI3K inhibitor LY294002 or MEK inhibitor PD98059 for 10 minutes, then treated with EPO for 5 minutes. Cytoplasmic extracts were subjected to Western blotting with anti–Raf-1 antibody. (B) Cytoplasmic extracts were subjected to Western blotting with anti–Raf-1 phosphoserine 338 antibody.