Ser70- or multisite phosphorylation of Bcl2 retards G1/S transition and cell growth in association with increased cell survival. (A) WT and A- or E-containing Bcl2 mutants were stably transfected in NSF.N1/H7 cells. Bcl2 protein expression levels were determined by Western blotting using Bcl2 antibody. (B) NSF.N1/H7 cells expressing WT or Bcl2 mutants were harvested under normal growth conditions. Cell cycle status was analyzed by flow cytometry following PI staining. (C) Cells expressing WT or A- or E-Bcl2 mutants were deprived of IL-3 or treated with VP-16 or adriamycin in the presence of IL-3 for 5 days. Cell viability was determined by analyzing annexin-V binding on FACS as described previously.17 (D) Growth curves of cells expressing WT and Bcl2 mutants were assessed using Coulter Counter. Similar results were obtained in all these studies using 3 separate clones each expressing similar amounts of exogenous Bcl2. Representative results for one clone are presented. The data represent the mean ± SD of 3 determinations.
