Activation of AKT by EPO is absolutely dependent on PI3K activity.

EPO was removed from UT-7/EPO cells for 24 hours. The cells were pretreated with increasing concentrations of LY294002 (10-100 μmol/L) and then stimulated with EPO (10 U/mL) for 10 minutes. 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.