14-3-3 prevents BAD dephosphorylation in vitro.
(A) FL5.12BCL-XL/BAD lysates were prepared in phosphatase buffer A with or without 1% empigen BB (Emp) (upper panel) or 25 μM R18 peptide (lower panel). BAD dephosphorylation was carried out for 30 minutes at 4°C or 30°C, followed by Western blotting for BAD. (B) BAD was immunoprecipitated by polyclonal C20 Ab from FL5.12BCL-XL/BAD cells in isotonic IP buffer with or without 1% empigen BB (Emp), in the presence or absence of 0.1 μM microcystin, and the immunocomplexes were analyzed by Western blotting. BAD was detected by monoclonal Ab 31420. The same blot was probed for 14-3-3 using polyclonal Ab K-19. (C) BAD was immunoprecipitated in isotonic buffer with or without 25 μM R18 peptide, in the presence or absence of 1 μM okadaic acid (OA), and analyzed by Western blotting as in panel B. (D) BAD was immunoprecipitated in isotonic buffer, R18 peptide was or was not added to the BAD immunocomplex, which was washed, and 40 μg FL5.12 lysate was added and incubated at 30°C in phosphatase buffer B. Reaction products were analyzed by Western blotting for BAD and 14-3-3 as in panel B.
