Figure 7
From: Starfish Apaf-1 activates effector caspase-3/9 upon apoptosis of aged eggs
Apoptosome-like complex formation by recombinant procaspase-3/9-His6 in the cell-free preparations. (a) Western blot analysis of recombinant GST-sfApaf-1 CARD (1–134 aa) protein using anti-sfApaf-1 antibody. Lanes: (1) without IPTG induction; (2) with IPTG induction at 37 °C. (b) Ultracentrifuged cell-free preparations were fractionated by gel filtration chromatography. Endogenous procaspase-3/9 and sfApaf-1 were detected by western blotting with anti-caspase-3/9 and anti-sfApaf-1 antibodies. (c) Activation of endogenous caspase-3/9 in cell-free preparations by treatment with procaspase-3/9-His6. A time course of DEVDase activity was measured at the indicated times after adding either procaspase-3/9-His6 or buffer (control). (d) Ultracentrifuged cell-free preparations were incubated with recombinant procaspase-3/9-His6, and fractionated by gel filtration chromatography. Fractions were analyzed by western blotting with the anti-caspase-3/9 antibody and anti-sfApaf-1 antibody. (e) DEVDase activity in fractions was measured by the cleavage of Ac-DEVD-MCA. The red column is from gel filtered cell-free preparations with procaspase-3/9-His6, and the blue column is from gel filtered cell-free preparations without recombinant protein. Full blots are presented in Supplementary Fig. S10. The results are representative of two independent experiments.
