Toso forms a trimolecular complex with RIP1 and FADD. (A) Toso constitutively associates with RIP1. Wild-type (lanes 1, 3, 4, 6, 7), RIP deficient (lanes 2,5) Jurkat cells or wt BJAB cells (lanes 8-11) were transiently transfected with a Toso-FLAG construct. Cell lysates from anti-hCD95 (CH11) treated and untreated cells were immunoprecipitated (IP) using anti-FLAG coupled agarose. The control IP of wt Jurkat cells using mouse Ig coupled agarose is shown in lane 1. Control or Toso immunoprecipitates and corresponding total cell lysates were analyzed by Western blotting for RIP1, FLAG and actin. Representative data from 3 independent experiments are shown. (B) RIP1 is required for the association of Toso with FADD. RIP deficient Jurkat cells (lanes 1-2) or wt Jurkat cells (lanes 3-4) were transfected with Toso-FLAG and stimulated with anti-CD95 (CH11) for 15 minutes or left untreated. Cell lysates were immunoprecipitated with anti-FLAG coupled agarose. Immunoprecipitates were analyzed by Western blotting for RIP1, FLAG or FADD. Data are representative of 3 experiments. (C) Toso and RIP1 ubiquitination promote the interaction of RIP1 with FADD. Jurkat cells stably expressing Toso miRNA (lanes 1-2), wt Jurkat (lanes 3-4), or Toso overexpressing Jurkat cells (lanes 5-8) were transfected with FLAG-RIP1 (lanes 1-6) or FLAG-K377R RIP1 (lanes 7-8). Untreated cells or cells stimulated with anti-CD95 (CH11) for 15 minutes were immunoprecipitated with anti-FLAG coupled agarose. Proteins bound to the RIP1 complexes were immunoblotted with antibodies against FADD (top) or FLAG (bottom). Data are representative of 2 experiments.