FIG. 4.

Phosphorylation of BCL-2 during the cell cycle. (A) Phosphorylation of endogenous BCL-2 at G₂/M phase. Jurkat-Neo cells were elutriated to enrich for G₁, S, and G₂/M fractions and compared to asynchronous cells (Cont). Cellular lysates were subjected to immunoprecipitation (IP) with anti-BCL-2 monoclonal Ab 6C8 and the subsequent Western blot was developed with anti-hBCL-2 Ab Bcl-2/100. The percentages of cells in G₁, S, and G₂/M were determined by PI staining using FACScan and CELLQUEST software. (B) Ser70 was phosphorylated in cycling cells. Jurkat clones expressing WT, S70A, or S87A BCL-2 were elutriated and subjected to Western analysis. (C) Cells with phosphorylated BCL-2 at G₂/M demonstrated increased susceptibility to apoptosis. Jurkat clones expressing WT or S70A BCL-2 were elutriated, and typical G₁-, S-, and G₂/M-enriched fractions were compared to asynchronous cells (Cont). Cells were treated with 100 ng of anti-Fas Ab per ml, and cell viability was determined by PI exclusion 6 h later. Values represent the relative viability of each fraction when the viability of the asynchronous cells is set at 100%. Values are the means of duplicate assays.