RIPK1S25D/S25D macrophages are protected against LPS/zVAD-fmk-induced necroptosis. Bone marrow-derived macrophages (BMDMs) were isolated from RIPK1+/+ and RIPK1S25D/S25D mice and treated with 50 ng/mL LPS and 20 µM zVAD-fmk for 18 h. (A) Cell death was measured using propidium iodide (PI) labeling. (B,C) Western blot analysis on cell lysates of necroptosis proteins (RIPK1, RIPK3, MLKL) as well as proteins associated with the scaffolding function of RIPK1 regulating inflammatory signaling (NF-κB, IκBα, p38). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA followed by Tukey’s post hoc test, n = 5 independent experiments).