Fig. 2

Interaction of α14 with RIPK3 is required for monitoring necroptosis. a Diagram of a series of α14 mutants. F234E; phenylalanine at 234 is substituted with glutamic acid, 4ST5A; S228, S345, T347, S349, and S352 were substituted with alanines. Zigzag lines indicate the regions that were replaced with SAGG repeats. Red boxes indicate each α helix. b L929 cells were transiently transfected with the indicated mutants and then stimulated with TZ, and the FRET/CFP ratio was analyzed as in Fig. 1b. Maximum changes of the FRET/CFP ratio. Each dot indicates individual cell (n = 10–14 cells). Statistical significance was determined using the one-way ANOVA test. *P < 0.05, ***P < 0.001, ns, not significant. c HEK293T cells were transiently transfected with the indicated mutants along with FLAG-RIPK3. Cell lysates were immunoprecipitated and analyzed as in Fig. 1c. Results are representative of two independent experiments. d GSK’872 treatment does not affect the binding of SMART to RIPK3. HEK293T cells were transiently transfected with SMART or 4ST5A along with FLAG-RIPK3. At 24 h after transfection, cells were untreated or treated with GSK’872 for 4 h, and cells were subjected to immunoprecipitation and analyzed as described in Fig. 1c. Results are representative of two independent experiments, or pooled results of two independent experiments (b). Error bars indicate s.e.m.