EPO induces serine phosphorylation of Akt protein in time- and dose-dependent fashions in UT-7/EPO cells.

EPO was removed from UT-7/EPO cells for 24 hours. The cells were then stimulated with EPO (10 U/mL) for the periods indicated (A), or with increasing concentrations of EPO (0.01-100 U/mL) for 10 minutes (B). After solubilization, cell lysates were immunoprecipitated with protein G-conjugated anti-Akt antibody. Immunoprecipitates were eluted with buffer containing SDS and resolved by 10% SDS-PAGE. Proteins were transferred onto a PVDF membrane. Upper panel: immunoblotting with antiphospho Akt antibody. Lower panel: the blot was reprobed with anti-Akt serum to confirm equal loading of protein.