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. 2004 Apr 22;23(10):2134–2145. doi: 10.1038/sj.emboj.7600210

Figure 7.

Figure 7

PHAPI enhances the catalytic activity of apoptosomes. (A) Caspase-9 apoptosome complexes (left) and Jurkat cell-free extracts (right), incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of 50 μg/ml cytochrome c/1 mM dATP, were assayed for DEVDase activity in the presence of 0.5 μM recombinant PHAPI (triangles), PHAPI-ΔTail (squares) or buffer alone (circles). (B) Caspase-9 apoptosome complexes (left) and Jurkat cell-free extracts (right), incubated for 15 min at 37°C in the presence (filled symbols) or absence (open symbols) of cytochrome c/dATP, were assayed for DEVDase activity in the presence of 0.5 μM recombinant PHAPI (small circles), GST (large triangles), GST-PHAPI-Tail (small triangles), 15–50 kDa polyglutamine polymers (squares) or buffer alone (large circles). The results shown are representative of three separate experiments. (C) Aliquots (750 ng) of purified bacterially expressed PHAPI, PHAPI ΔTail, GST and GST-PHAP-Tail proteins were separated by SDS–PAGE, and visualized by coomassie blue staining. A degradation product of full-length PHAPI is indicated by an asterisk. (D) Jurkat cell-free extracts, incubated for 30 min at 37°C in the presence (filled symbols) or absence (open symbols) of 2 μg/ml cytochrome c/1 mM dATP, were assayed for DEVDase activity in the presence of 1 μM recombinant PHAPI (small circles), 5 μM okadaic acid (triangles), 1 μM calyculin A (squares) or buffer alone (large circles).