Figure 3.

Loop deletion mutant of Bcl-2 blocks paclitaxel-induced Bcl-2 phosphorylation and apoptosis. MDA-MB-231 cells were transiently transfected with a total of 2 μg/ml DNA of either empty vector (−) or Bcl-2 (WT, S70A, S51A, S87A, S105A, ΔTM, or Δloop) expression plasmid with Lipofectamine and incubated for 1 day. Cells were washed, and fresh medium was added. After 8 h, the cells were incubated with 100 nM paclitaxel (+) or vehicle (−) for 4 h and 24 h for the measurements of Bcl-2 phosphorylation (A) and apoptosis (B), respectively. Cells were stained with 4′,6-diamidino-2-phenylindole to examine apoptotic nuclei. (C) Phosphorylated Bcl-2 is dephosphorylated by PPase I, but not by PTPase in MDA-MB-231 cells. Cells were transfected with Bcl-2 (WT and mutants) as described above and treated with paclitaxel (100 nM) for 4 h. Cell lysates then were incubated with either PPase I (5 μM) or PTPase (5 μM).