Blockage of Smac/DIABLO with antibodies inhibits cytochrome c release and caspase-3 activation.

<p>A. Smac/DIABLO antibodies reduce osmostress induced apoptosis. Oocytes were injected with anti-Smac/DIABLO (25 or 50 ng/μl final concentration) or with anti-human IgG (50 ng/μl) as a control, and treated with 300 mM sorbitol. Cytosolic extracts were obtained at different times to measure Smac/DIABLO, cytochrome c (CC), cleaved caspase-3, fodrin α, cleaved fodrin α, and actin (loading control) by Western blot. The result is representative of three independent experiments. B. Smac/DIABLO antibodies interact with Smac/DIABLO in cytosolic fractions. Oocytes were treated with 300 mM sorbitol and cytosolic extracts were obtained at 3 h and incubated with anti-Smac/DIABLO from ProSci (Ab1), anti-Smac/DIABLO from Calbiochem (Ab2), or rabbit IgG from Sigma as a control antibody. 100 μl of cytosolic extract were incubated with 5 μl of the corresponding antibody (1μg/μl) for 2 h at 4°C on a rotating wheel before their analysis by native-PAGE or SDS-PAGE and Western blot to measure the levels of Smac/DIABLO, AMPK, and the antibodies. C. Smac/DIABLO antibodies interact in vivo with Smac/DIABLO. Oocytes were injected with Ab1, Ab2, or rabbit IgG (50 ng/μl final concentration), and treated with 300 mM sorbitol for 3 h. Cytosolic extracts were analyzed by native-PAGE or SDS-PAGE and Western blot to measure the levels of Smac/DIABLO, AMPK, and the antibodies.