FIG. 3.
Dephosphorylation of pSer112 precedes and promotes pSer136 dephosphorylation during survival factor withdrawal. (A) Lysates of FL5.12 BCL-XL/BAD cells at the indicated times of IL-3 withdrawal were immunoblotted for pSer112, pSer136, pSer155, and total BAD (31420; Santa Cruz). Representative immunoblots are shown. Percentages of phosphorylation indicate the averages of pSer residue phosphorylation of two to three experiments as measured by densitometry. The ratio of each pSer residue phosphorylation to total BAD at time zero was set as 100%. (B) FL5.12 cells expressing the 112A BAD mutant were immunoblotted for pSer136 and compared to cells expressing wild-type (WT) BAD during IL-3 withdrawal. Percent phosphorylation was quantitated as in A. (C) Lysates of FL5.12 BCL-XL/BAD cells collected at the indicated times after IL-3 withdrawal were allowed to dephosphorylate in the absence or presence of R18 and immunoblotted for pSer136 and total BAD. (D) Graph of relative pSer136 dephosphorylation in the absence of R18, shown in C, as measured by densitometry. The ratio of pSer136 to total BAD at time zero was set as 100%. Percent pS136 phosphorylation is the ratio of pSer136 to total BAD after 20 min at 30°C in the absence of R18 compared to that at time zero in three experiments.