Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex

CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor n ecrosis factor receptor superfamily. Multimerization of CD95 leads to insta nt recruitment of the signaling molecules FADD and caspase-8 to the activat ed receptor forming the death-inducing signaling complex (DISC). DISC forma tion is the first essential step of CD95 signaling and results in activatio n of caspase-8 starting a signaling cascade that leads to apoptosis. Here w e describe a method for analyzing the CD95 DISC. The method is based on coi mmunoprecipitation of the signaling molecules with the activated CD95 recep tor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95-mediate d apoptosis. (C) 1999 Academic Press.