Molecular targets of PTEN. (A) WM35 (lanes 1 and 2), A375-S2 (lanes 3 and 4), and MeWo (lanes 5 and 6) cells were transduced with either Ad-Luc (odd lanes) or Ad-PTEN (even lanes). Twenty-four hours postinfection, cells were harvested, lysed, and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and probed with antibodies against PTEN, AKT/PKB, Phospho-AKT, p27, Apaf-1, and actin. (B) AKT kinase activity is inhibited by Ad-PTEN. Mock treated, Ad-Luc-, or Ad-PTEN-treated MeWo cells were extracted and their AKT kinase activity was measured by 32P-labeling using GS3K as substrate. (C) The level of phospho-specific AKT/PKB decreased following Ad-PTEN transduction. MeWo cells were mock infected or infected with Ad-PTEN or Ad-Luc and harvested after 24 or 48 hr following infection. Total cell lysates were prepared and probed for p-AKT. The treatment of cells with LY 294002 was used as a positive control for AKT/PKB dephosphorylation.