RIPK1 R606me2s suppresses RIPK1 kinase activation and the formation of complex I and complex II to inhibit subsequent cell death. (A and B) MEFs were reconstituted with WT or R606K mutant RIPK1 (A) or treated with negative control siRNAs or siRNAs targeting ADK (B). The cells were stimulated by Flag-TNFα (100 ng/ml) after pretreatment of 5z7 (500 nM) for 30 min. After stimulation for indicated periods, complex I was immunoprecipitated with Flag antibody. Relative levels of TNFR1, p-RIPK1 (S166), RIPK1, TRADD in complex I or whole-cell lysates were measured with immunoblotting. (C and D) MEFs were reconstituted with WT or R606K mutant RIPK1 (C) or treated with negative control siRNAs or siRNAs targeting ADK (D). The cells were stimulated by Flag-TNFα (10 ng/ml) after pretreatment of 5z7 (500 nM) for 30 min. After stimulation for indicated periods, RIPK1 was immunoprecipitated. Relative levels of RIPK1, FADD, TRADD in RIPK1 immunocomplex or whole-cell lysates were measured with immunoblotting. (E and F)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were subsequently treated with 5z7 (500 nM)/TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (F). (G and H)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were subsequently treated with CHX (C, 2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα (T) for the indicated time in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-S166 RIPK1, p-T231/S232 RIPK3, and p-S345 MLKL were determined by immunoblotting (H). Data are represented as the mean ± SD (E and G). Data are representative of n = 3 independent experiments (A–H). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (E and G). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F5.
Figure 5.

RIPK1 R606me2s suppresses RIPK1 kinase activation and the formation of complex I and complex II to inhibit subsequent cell death. (A and B) MEFs were reconstituted with WT or R606K mutant RIPK1 (A) or treated with negative control siRNAs or siRNAs targeting ADK (B). The cells were stimulated by Flag-TNFα (100 ng/ml) after pretreatment of 5z7 (500 nM) for 30 min. After stimulation for indicated periods, complex I was immunoprecipitated with Flag antibody. Relative levels of TNFR1, p-RIPK1 (S166), RIPK1, TRADD in complex I or whole-cell lysates were measured with immunoblotting. (C and D) MEFs were reconstituted with WT or R606K mutant RIPK1 (C) or treated with negative control siRNAs or siRNAs targeting ADK (D). The cells were stimulated by Flag-TNFα (10 ng/ml) after pretreatment of 5z7 (500 nM) for 30 min. After stimulation for indicated periods, RIPK1 was immunoprecipitated. Relative levels of RIPK1, FADD, TRADD in RIPK1 immunocomplex or whole-cell lysates were measured with immunoblotting. (E and F)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were subsequently treated with 5z7 (500 nM)/TNFα (10 ng/ml) for the indicated time (E) or 12 h (F) in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (E). The levels of p-RIPK1(S166), CC8, and CC3 were determined by immunoblotting (F). (G and H)Ripk1−/− MEFs were reconstituted with WT or R606K mutant Flag-RIPK1 by lentivirus. The cells were subsequently treated with CHX (C, 2 μg/ml) and zVAD.fmk (Z, 10 μM) for 0.5 h followed by 10 ng/ml TNFα (T) for the indicated time in the presence or absence of Nec-1s (10 μM). Cell death was measured by the SYTOX Green positivity assay (G). The levels of p-S166 RIPK1, p-T231/S232 RIPK3, and p-S345 MLKL were determined by immunoblotting (H). Data are represented as the mean ± SD (E and G). Data are representative of n = 3 independent experiments (A–H). Statistical significance was determined using two-way ANOVA with post hoc Bonferroni’s test (E and G). **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F5.

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