STAT1-deficient erythroblasts reveal elevated phosphorylation of STAT5a, STAT5b, PKB, and Erk1/2. Wild-type and STAT1^–/– splenic proerythroblasts were depleted of cytokine for 6 hours and then stimulated with increasing concentrations of EPO for 15 minutes. Following cell lysis, an immunoprecipitation was performed with peptide-specific antibodies against STAT5a (A) or STAT5b (B). Immune complexes were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an antiphospho-STAT5 antibody. The membrane was stripped and reprobed with a peptide-specific antibody recognizing total STAT5. (C) Lysates (100 μg) were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an antiphospho-ERK1/2 monoclonal antibody. The membrane was stripped and reprobed with an antibody that recognizes total ERK1/2. (D) Lysates (100 μg) were resolved by SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with an antiphosphoserine-PKB polyclonal antibody. The membrane was stripped and reprobed with an antibody that recognizes total PKB.