Fig. 6. JNK is recruited to necrosome and then regulates necroptosis through RIPK1 and TRIF.

a, b Peritoneal macrophages were treated with TNF+zVAD (a) or poly I:C+zVAD (b) for the indicated time. Cell lysates were immunoprecipitated with anti-RIPK1 antibody and then analyzed by immunoblotting using the indicated antibodies. c Peritoneal macrophages were pretreated with zVAD, DMSO, or SP600125 for 30 min, followed by TNF, poly I:C, or LPS treatment for 3 h. Lysates were analyzed by immunoblotting with indicated antibodies. d Immunoblot analysis of peritoneal macrophages transfected with indicated siRNA for 3 days and then treated with TNF+zVAD for 3h. e Peritoneal macrophages were transfected with indicated siRNA for 3 days and then treated with TNF+zVAD and poly I:C+zVAD for 6h. Cell death was measured by released LDH. f Peritoneal macrophages transfected with control siRNA and JNK-specific siRNA for 3 days and then the expression of TLR3, TLR4, Myd88, IRAK4, A20, cyld, BIRC2, BIRC3, cFlip, JNK1, and JNK2 determined via real-time PCR. g, h Immunoblot analysis of TRIF in peritoneal macrophages (g) or MEF cells (h) transfected with indicated siRNA for 3 days. i Immunoblot analysis of TRIF in peritoneal macrophages transfected with indicated siRNA for 3 days, and then treated with TNF+zVAD or poly I:C+zVAD for 3 h. Data are representative of at least three independent experiments and shown as mean±SEM in graph e, f. *p < 0.05 by Student’s t test. NS no significance, LE long exposure, SE short exposure