Okadaic acid inhibits BAD dephosphorylation.
(A) FL5.12BCL-XL/BAD cells were treated with 0.5 μM OA or dimethyl sulfoxide (DMSO) for 4 hours in the presence (+) or absence (−) of IL-3. Cell lysates were analyzed by Western blotting. The blot was first probed with monoclonal anti-BAD Ab 31420 (upper panel), then reprobed with antiphospho serine 112 BAD Ab (lower panel). pBAD, phosphorylated BAD. (B) FL5.12BCL-XL/BAD (wt BAD) and cells expressing the phosphorylation-defective BAD mutant (BAD112A136A) were treated with 0.5 μM OA with (+) or without (−) IL-3 for 3 hours, and analyzed by Western blotting using polyclonal anti-BAD Ab C20. (C) FL5.12BCL-xL/BAD cells were treated with indicated doses of OA in the presence of IL-3 for 3 hours and analyzed by Western blotting using anti-BAD Ab 31420. (D) Cells were treated with 0.5 μM OA for the times indicated and analyzed by Western blotting as above. (E) BAD phosphorylation induced by OA is the result of inhibition of BAD phosphatase activity, not indirect activation of kinases. FL5.12BCL-XL/BAD cells were grown in the presence (lanes 1, 5) or absence of IL-3 (lanes 2-4) for 3 hours. Cells in lanes 3 to 5 were then treated with DMSO (lane 3) or 0.5 μM OA (lanes 4, 5) for another 3 hours. Lysates were analyzed by Western blotting as above.
