Figure 9

AAV2 induction of apoptosis/cell death in MDA-MB-231 cells results in activation of caspases of both the intrinsic and extrinsic pathways but not PARP cleavage. Left panel: MDA-MB-231 monolayer cell cultures were synchronized in G1, followed by infection with AAV2. Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of caspases and their cleavage/activation was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control MDA-MB-468 cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect the 35 kDa pro-caspase form of caspase-3, proteins were resolved in a 10% SDS-PAGE gel and detected with caspase-3 rabbit monoclonal antibody (Cell Signaling Technology). To detect the17-kDa cleaved caspase-3 forms, proteins were resolved in a 15% SDS-PAGE gel and detected with rabbit polyclonal antibody against cleaved caspase-3 (Cell Signaling Technology). To detect the 35 kDa pro-caspase form of caspase-6, proteins were resolved in a 10% SDS-PAGE gel and to detect the 15 kDa cleaved form of caspase-6, proteins were resolved in a 15% SDS-PAGE gel and detected with a rabbit polyclonal antibody (Cell Signaling Technology). To detect both the pro- and cleaved forms of caspase-7, caspase-8 and caspase-9, proteins were resolved in a 10% SDS-PAGE gel. The 35 kDa pro-caspase form and the 30 kDa/20 kDa cleaved forms of caspase-7 was detected with a mouse monoclonal antibody (Cell Signaling). The pro-caspase and cleaved 28 kDa form of caspase-8 was detected with a mouse monoclonal antibody (Alexis Biochemicals). The 47 kDa pro-caspase and 37 kDa/35 kDa cleaved forms of caspase-9 were detected with a rabbit polyclonal antibody (Cell Signaling). To detect the pro- (116 kDa) form of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).