Akt stimulates HIF-1α stabilization. (A) U373 cells were retrovirally infected with wild-type PTEN. Thirty-six hours postinfection, U373 parental cells and PTEN-expressing U373 were subjected to hypoxia for the indicated time. Cells were lysed at the indicated times followed by SDS-PAGE, transfer, and immunoblotting with anti-HIF-1α. (B) Cells were retrovirally infected with myr–Akt–ER or myr–A2–ER. Thirty-six hours postinfection, the cells were subjected to induction by 4-HT for the indicated time and analyzed as in A. (C) U373 cells were serum-deprived and subjected to 1 hr of hypoxia to activate Akt or to UV-C (10 J/m2) for JNK-1 activation for use in immune complex kinase assays. Reactions were performed using 500 ng of either histone H2B, GST–jun, GST–GSK-3β, or HIF-1α as substrates and Akt or JNK-1 immunoprecipitations for in vitro kinase assays. Kinase reactions were subjected to SDS-PAGE, and gels were dried and visualized by PhosphorImaging. (D) Cells were subjected to hypoxia for the indicated times, lysed, immunoprecipitated using anti-HIF-1α, and subjected to SDS-PAGE, transfer, and immunoblot using anti-HIF-1α anti-Akt, or anti-HIF-1β antibodies.