FIG. 4.

PP2A competes with 14-3-3 to associate with BAD. (A) BAD was immunoprecipitated (IP) from FL5.12 cells or NIH 3T3 cells expressing vector or wild-type BAD in the presence of R18 and immunoblotted for the PP2A/cα and PP2A/Aα subunits of PP2A. (B) BAD was immunoprecipitated from NIH 3T3/BAD lysates in the absence (−) and presence of the 14-3-3 displacement reagents Empigen BB, R18 peptide, and pSer136 peptide and immunoblotted for PP2A/c, 14-3-3, and BAD. (C) Recombinant GST-BAD and in vitro protein kinase A-phosphorylated GST-BAD was incubated with NIH 3T3 lysates in the absence and presence of R18. Glutathione pulldowns were immunoblotted for 14-3-3 and PP2A/c as indicated. (D) Recombinant GST-PP2A/A and GST-PP2A/c were incubated with lysates from control NIH 3T3 cells or cells expressing wild-type BAD, and glutathione pulldowns were immunoblotted for BAD. Input recombinant GST and GST fusion proteins were detected with anti-GST, anti-PP2A/A, and anti-PP2A/c antibodies. (E) FL5.12 cells expressing GST-BAD were deprived of IL-3 for 30 min. GST-BAD was pulled down by glutathione-agarose in the absence (−R18) or presence (+R18) of R18 and immunoblotted for GST-BAD, PP2A/Cα, PP2A/Aα, and 14-3-3. One tenth of the total lysates from FL5.12 (FL) and FL5.12/GST-BAD (FL/GST-BAD) cells used for pulldown assays was loaded as a control.