RIP1 associates with caspase-8 and is required for its activation. (A and B) RIP1 knockdown suppresses CD154-induced caspase-8 cleavage in EJ (A) or HeLa/CD40mT6 (B) cells. Lysates were immunoblotted for caspase-8 or, as a control, β-actin or p50 NF-κB. (C) RIP1, TRAF2, FAF1, and caspase-8 interact upon CD40 stimulation. Cells were stimulated with CD154, and lysates were immunoprecipitated with a goat polyclonal against caspase-8. Immunoprecipitants were immunoblotted using rabbit polyclonal antibodies against RIP1, TRAF2, CD40, and FAF1 or a monoclonal anti-caspase-8, as indicated. Results are representative of three independent experiments. The Ig light chains are indicted by asterisks. (D and E) Expression of FADD (D) and FAF1 (E) before and after transfection with the respective siRNAs. (F and G) Crystal violet staining of RNAi-transfected EJ cells treated with CHX in the presence or absence of CD154 or CD95L. (H) FAF1 but not FADD knockdown protects from the pro-apoptotic effects of CD40 ligation. After knockdown, EJ cells were exposed to CD154 and CHX for 5 h before assessment of apoptosis. Error bars indicate SD. (I) Proposed model of CD40-induced death signaling in tumor cells. CD40-mediated apoptosis involves the formation of a secondary cytoplasmic complex of TRAF2, RIP1, FAF1 and caspase-8 (Casp-8). RIP1 is required for caspase-8 activation and cell death, whereas apoptosis is antagonized by survival signals predominantly mediated by the TRAF6-binding domain of CD40, which may operate at the level of the death-inducing signaling complex and/or downstream of it.