Fig. 5. Carboxyl-terminal caspase-8 mutants efficiently dimerize with WT caspase-8 but are deficient in mediating TRAIL-induced apoptosis, while amino-terminal mutants fail to dimerize but are differentially effective at mediating apoptosis.

Co-immunoprecipitations were performed to detect heterodimerization between MT caspase-8 proteins and WT caspase-8. A HeLa-CASP8 KO cells engineered for DOX-inducible expression of WT caspase-8-FLAG were treated in the absence or presence of DOX. Simultaneously, the cells were transiently transfected with constructs encoding the indicated HA-tagged caspase-8 mutants. Eight hours after transfection, cell lysates were prepared and subjected to immunoprecipitation with anti-FLAG, followed by immunoblotting with anti-HA to detect dimerization (for immunoprecipitations: 500 μg of input for D303G, L62P, R248W, T272del, D303V, T441I, R465*; 2000 μg of input for L7v, G11E, G11R, R71T, L105H, Y178del, D308G, R435*; 125 μg of input for S375* and S386*; 1750 μg of input for S99F). B Input lysates were analyzed by immunoblotting for expression of the MT caspase-8-HA and WT caspase-8-FLAG proteins. Due to varying levels of expression of the HA-tagged MT proteins, varying amounts of input lysate were loaded (25 μg for D303G, L62P, R248W, T272del, D303V, T441I, R465*; 100 μg for L7V, G11E, G11R, R71T, L105H, Y178del, D308G, R435*; 12.5 μg for S375* and S386*; 87.5 μg for S99F) Experiments in panels A and B were performed twice times with similar results. C The figure shows an inverse relationship for the caspase-8 mutants between the ability to mediate TRAIL-induced cell death and the capacity to heterodimerize with WT caspase-8.