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Figure 7 | Molecular Cancer

Figure 7

From: Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

Figure 7

AAV2 induction of apoptosis in MCF-7 cells results in PARP cleavage independent of caspase activation. MCF-7 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 MCF-7 and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect the 35 kDa pro-caspase form of caspase-6, proteins were resolved in a 10% 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 of caspase-7 was detected with a mouse monoclonal antibody (Cell Signaling). The 58 and 56 kDa forms of caspase-8 were detected with a mouse monoclonal antibody (Alexis Biochemicals). The 47 kDa pro-caspase form of caspase-9 was detected with a rabbit polyclonal antibody (Cell Signaling). To detect the pro- (116 kDa) and cleaved- (89 kDa) forms of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).

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