BAD protein phosphorylation by the PI3′K/PKB pathway.

DP16.1/p53ts cells were left untreated or treated with LY294002 (5 μM) for 60 minutes. Cell extracts were prepared and analyzed by 2-dimensional gel electrophoresis followed by Western blot analysis as described in “Materials and methods.” A portion of the untreated DP16.1/p53ts extract was incubated with λ-phosphatase (20 U/μL) prior to isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. BAD proteins were visualized by immunoblotting with antibodies against BAD or with phospho-specific antibodies recognizing Ser112-phosphorylated BAD.