Raf-1 undergoes a mobility shift in response to EPO, which can be reversed by phosphatase treatment. (A) BaF3-EpoR cells or normal murine splenic erythroid cells were treated with EPO for 5 minutes, and cytoplasmic extracts were subjected to Western blotting with anti–Raf-1 antibody. Note mobility shift (hypershift) Raf-1(H). (B) BaF3-EpoR cell cytoplasmic extract was incubated with phosphatase PTP-1B or PP2A for 20 minutes at 30° C, followed by Western blotting with anti–Raf-1 antibody. (C) The membrane shown in panel B was stripped and reprobed with anti–Raf-1 phosphoserine 338 antibody. (D) Cytoplasmic extract was incubated with a combination of phosphatases PTP-1B and PP2A for 20 minutes at 30° C, followed by Western blotting with anti–Raf-1 antibody.