TCTP interacts with CARD of Apaf-1 to form an apoptosome complex. (A) Binding of TCTP to Apaf-1 in apoptosome-forming condition. TCTP, procaspase-9, and cytochrome c binding to Apaf-1 was analyzed. Recombinant human TCTP, cytochrome c, dATP and S-100 extract were incubated, immunoprecipitated with anti-Apaf-1-specific antibody and then blotted with antibodies, as described in Methods. (B) Interaction of TCTP with CARD of Apaf-1. Schematic diagram of full-length Apaf-1 and its variants devoid of particular domain(s) was presented. The full-length Apaf-1 (residues 1-1194) contains CARD (residues 1-97), CED-4 (residues 98-412), and WD-40 repeats (residues 413-1194). Recombinant Full APAF-1, APAF-530 (residues 1-530), APAF-420 (residues 1-420), APAF-97 (residues 1-97), and TCTP-GST (GST-tagged TCTP) were constructed. Purified WDR (WD Repeat) protein lacking both CARD and CED-4 was obtained from Abnova Corporation. Binding of TCTP-GST and His-tagged Apaf-1 constructs was analyzed using pull-down assay. Protein binding complexes were separated on SDS-PAGE and stained with silver. Purified TCTP-GST protein was also resolved by SDS-PAGE for detection of TCTP (last lane). NC, negative control.