TUCAN does not interact with procaspase-9 or Apaf-1. Immunoprecipitation of in vitro translated procaspase-9 (procaspase-9 mut) mixed with a FLAG-tagged version of TUCAN (TUCAN-FLAG) (A), and procaspase-9 mixed with a truncated variant of Apaf-1 (Apaf-1 (1–570)) as positive control (B). Immunoprecipitation was performed with anti-caspase-9- or anti-Flag antibodies. The total of radiolabelled proteins in the in vitro-translation mixture prior to immunoprecipitation is also included (total). A low molecular weight band obtained in the total TUCAN mixture that did not react with the anti-Flag antibody is indicated with an asterisk. C) H460 cell extract was subjected to caspase-9 pull-down in the presence (+) or absence (-) of exogenously supplied cytochrome c and dATP followed by western blot detection of caspase-9 and TUCAN, or Apaf-1 as positive control. As a control, total cell extract (total) was included.