Akt activity regulates Bcl-w expression.

<p>(A) HeLa cells were transfected with 2 µg of HA-Akt wt, Akt D+, or HA-Akt D− cDNA and 2 µg Flag-Bcl-w for 48 hrs. Protein extracts were immunoprecipitated with an anti-HA monoclonal antibody. Immunoprecipitates were resolved on 12% SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with anti-Flag antibody (0.2 µg/ml). 50 µg of total sample extracts were also analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (B) HeLa cells were transfected with 4 µg of HA-Akt wt, HA-Akt D+, or HA-Akt D− cDNA for 48 hrs. Protein extracts were blotted with anti-Bcl-w antibody in order to detect endogenous levels of Bcl-w. Loading control was obtained with anti-β actin antibody. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cellular proteins were solubilized and analyzed by western blot using the indicated antibodies. (D) HeLa cells were treated with 10, 20 or 40 µM of LY294002 for 24 hrs. Protein extracts were analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (E) Bcl-w HeLa cells were treated with 10 µM of MG-132 for 8 hrs. 40 µg of protein extracts were analyzed by western blot with anti-Bcl-w antibodies. Loading control was obtained using anti-β actin antibody.