Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c <t>(hcytc-</t> His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM <t>dATP</t> and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
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