PP2A-selective inhibitor, but not PP1-specific inhibitor, inhibits BAD dephosphorylation in vitro.

(A) FL5.12BCL-X_L/BAD lysates were prepared in phosphatase buffer A with or without fostriecin, in the presence or absence of 25 μM R18 peptide, incubated at 30°C for 20 minutes, and analyzed by Western blotting—first for BAD with monoclonal Ab 31420 (upper panel) and then reprobed with polyclonal antiphosphoserine 112 Ab (lower panel). (B) PP2A dephosphorylated BAD in vitro. Immunoprecipitated BAD was incubated with purified PP2A and analyzed by Western blotting. (C) FL5.12 lysates with or without the addition of 1.6 μM of the PP1 inhibitor I-2 were used as the source of phosphatase to react with immunoprecipitated BAD. (D) FL5.12 lysates preincubated with 1.6 μM inhibitor I-2 for 15 minutes without (I-2) or with the addition of 5 nM OA (I-2+OA 5 nM) were assayed for phosphatase activity using ³²P-labeled phosphorylase a as substrate. Results were expressed as a percentage of ³²P release in lysates with no additions (CTL) and represent mean ± SD of triplicate assays.