Figure 6 | Scientific Reports

Figure 6

From: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs

Figure 6

Activation of caspase-3/9 by sfApaf-1 CARD. (a) Activation of endogenous caspase-3/9 in cell-free preparation by GST-A-CARD. A time course of DEVDase activity was measured at the indicated time after addition of GST-A-CARD or control GST. (b) Cleavage of endogenous caspase-3/9 in cell-free preparations was induced by GST-A-CARD but not by GST, and detected by western blotting with the anti-caspase-3/9 antibody. (c) The interaction between A-CARD and caspase-3/9 in cell-free preparations. The cell-free preparation was incubated with GST-A-CARD or not, and fractionated by using gel filtration chromatography. Each fraction was analyzed by western blotting with anti-caspase-3/9 and anti-GST antibodies. (d) DEVDase activity in each fraction was measured by the cleavage of Ac-DEVD-MCA. The yellow column is from gel filtered cell-free preparations incubated with GST-A-CARD, and the blue column is from gel-filtered cell-free preparation without recombinant protein. Full blots are presented in Supplementary Fig. S10. The results are representative of three independent experiments.