Interaction of sfApaf-1 CARD with caspase-3/9 CARD. (a) Glutathione Sepharose 4B bead-bound GST-A-CARD was incubated with His-C-CARD in PBS buffer; glutathione Sepharose 4B-bound GST was used as a control. Associated His-C-CARD was co-eluted with GST-A-CARD (right panel), but not with GST (left panel) after the addition of elution buffer, as determined by SDS–PAGE and CBB gel staining. (b) Glutathione magnetic agarose bead-bound GST-A-CARD or similarly bead-bound GST (control) was incubated with cell-free preparations containing endogenous procaspase-3/9. Endogenous procaspase-3/9 was pulled down with GST-A-CARD (right panel), but not with GST (left panel), and identified by western blotting with anti-caspase-3/9 antibody. Full blots are presented in Supplementary Fig. S10. The results are representative of two independent experiments.