Figure 3
Prevention of apoptosis in MCF-7 cells by Bcl-2 and Bcl-XL and by mutants with restricted subcellular localization. A) Cell lysates containing 20 μg of protein from vector control (neo) or transfected MCF-7 cells expressing wild type proteins or proteins with restricted subcellular localization [ER (Bcl2-cb5, BclX-cb5), mitochondria (Bcl2-acta, BclX-acta) or nucleo-cytoplasm (BclX-Δt)] as indicated above the lanes were immunoblotted for PARP and actin. Cells were exposed to doxorubicin (Dox), TNFα and cycloheximide (TNF), ceramide (C2), or thapsigargin (TG) as in Figure 1. Untreated vector transfected cells (-). Expression of Bcl-2, Bcl-XL and the targeted mutants does not change the amount of PARP in untreated cells. The migration position of the 89 kDa caspase cleaved PARP fragment is indicated as ΔPARP. B) MCF-7 cells either untreated and transfected with the control plasmid (Neo -) or stably expressing the indicated protein and treated with TNFα and cycloheximide were stained with Hoescht 33342 (blue) or Annexin V coupled to Alexa Fluor594 (red) and visualized by epifluorescence microscopy. The width of each image is ~660 μm.