Fig. 7
A role for caspase-3-mediated DFF45 cleavage in DNA degradation in vitro. Protein extracts were isolated from sham operated or injured rat cortex isolated 72 hr after TBI. Sham samples were treated with indicated amounts of the recombinant rat caspase-3 for 1 hr at 37°C in the extraction buffer (20 mM Hepes, pH7.4, 2 mM EDTA, 1.5 mM MgCl2, 5 mM DTT, and 0.1% CHAPS). (A) Half of each extract was then analyzed by Western blotting using anti-DFF45 antibodies that recognize two alternatively spliced forms of DFF45 (DFF45L and DFF45S), their cleavage products (11 kDa), and a nonspecific band (16 kDa). (B) The other half of the extracts were incubated with rat cerebellar nuclei as described in Materials and Methods. The DNA was then analyzed by electrophoresis through a 1.5% agarose gel and ethidium bromide staining. The result demonstrates that cleavage of DFF45 by caspase-3 is not sufficient for DNA fragmentation induced by protein extracts from control rat brain cortex. Data are representative of four experiments with similar results.