Activated form of Akt prevents cell death induced by withdrawal of EPO.

(A) Generation of transfectant cells expressing HA-tagged Akt. The transfectant cells and the parent cells were harvested for detection of HA-tagged Akt proteins. Cell lysates were resolved by 10% SDS-PAGE. Proteins were transferred onto a PVDF membrane and immunoblotted with anti HA-antibody. (B) EPO was removed from the transfectant cells for 24 hours. Then the cells were stimulated with EPO (10 U/mL) for 10 minutes. After solubilization, cell lysates were immunoprecipitated with protein G-conjugated anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 10 μCi of [γ-³²P]ATP (3000 Ci/mmol) using histone H2B as a substrate (0.1 mg/mL). The reactions were incubated at 25°C for 30 minutes. Reaction products were resolved by 15% SDS-PAGE and visualized by autoradiography. (C) EPO was removed from the culture medium and the cell viability was assessed by trypan dye exclusion during the observation periods. (D) EPO was removed from the culture medium and the transfectant cells were cultured without any growth factors (shade). Three days later, cells were harvested and stained with annexin-V-FITC for detection of apoptotic cells (Apo) using FACScan. The cells cultured with EPO (1 U/mL) were used as a negative controls (light).