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Figure 2 Activation of mitochondrial death agonists. Neonatal rat ventricular cardiomyocytes were exposed to anti-erbB2 antibody (erbB2 Ab) for the specified times. Lysates were collected and separated by electrophoresis under denaturing conditions with protein detection by immunoblotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (a) Time-dependent increase in expression of bcl-associated protein. (b) Treated cells were subjected to subcellular fractionation to yield a mitochondria rich heavy membrane fraction (HM). The HM was incubated for 30 min with the irreversible crosslinker BMH. Immunoblot demonstrates crosslinked BAX multimers. (c) (Upper panel) Depletion of cytochrome c from the mitochondrial fraction, beginning at 8 hours after anti-erbB2 treatment. (Lower panel) Cytochrome oxidase used for control of mitochondrial protein loading. (d) Anti-erbB2 induced a time-dependent increase in cleavage and activation of caspase 9 beginning at 16 h. (e) Increase cleavage and activation of caspase 3 were also observed beginning as early as 8 h after treatment. (Bottom panel) Actin immunoblotting to control for cytosolic protein loading. All Western blots shown are representative of three or more independent experiments.