Fig. 5. TRIF-dependent cleavage of N4BP1 by caspase-8.

a–d Immunoblot analysis of N4BP1 in wild-type, Irak4^{otiose/otiose}, or Trif^lps2/lps2 peritoneal macrophages stimulated with LPS for the indicated times (a), in wild-type peritoneal macrophages stimulated with Pam3CSK4 (40 ng/ml), R848 (20 ng/ml), or poly(I:C) for the indicated times (b), or in wild-type peritoneal macrophages without or with siRNA-mediated caspase-8 knockdown and pre-treated with Z-VAD-FMK (20 μM), Necrostatin-1 (50 μM), or Z-IETD-FMK (50 μM) for 1 h and then stimulated with LPS for the indicated times (c, d). 40 pM siCaspase-8 was transfected with Lipofectamine 2000 into the cells 36 h before LPS stimulation (d). e TNF concentration in the culture medium of wild-type (WT), N4bp1^−/−, Trif^lps2/lps2N4bp1^−/−, or Trif^lps2/lps2 peritoneal macrophages treated with LPS at different concentrations (n = 3 mice per genotype). ****P < 0.0001, **P = 0.0054, two-way ANOVA and post hoc Tukey test. f Immunoblot analysis of HA-N4BP1 or truncation or point mutants of HA-N4BP1 expressed with or without TRIF in HEK293T cells. Asterisks indicate cleaved fragments of N4BP1. g Detection of the interaction between HA-N4BP1 or truncation mutants of HA-N4BP1 and Flag-NEMO expressed in HEK293T cells, assessed by immunoprecipitation with anti-HA followed by immunoblot analysis with anti-Flag. h Above, NF-κB-dependent luciferase reporter activity in wild-type or N4BP1^−/− HEK293T cells transfected with empty vector (−) or vectors encoding the indicated N4BP1 truncation mutants and stimulated with IL-1β or left unstimulated (none). n = 3 independent transfections of three cultures split from one cell line. Below, Immunoblot analysis of HA-N4BP1 or truncation mutants in the pooled, analyzed cells. *P = 0.0138, ***P = 0.0002, **P = 0.0022, **P = 0.0064, *P = 0.0225, *P = 0.0304, *P = 0.0447, unpaired, two-tailed Student’s t test. i NF-κB-dependent luciferase reporter activity in wild-type or N4BP1^−/− HEK293T cells transfected with empty vector (−) or a vector encoding the indicated N4BP1 mutant and stimulated with IL-1β, TNF, or left unstimulated (none). n = 3 independent transfections of three cultures split from one cell line. FL full-length N4BP1. **P = 0.0032, ***P = 0.0008, ****P < 0.0001, *P = 0.0343, unpaired, two-tailed Student’s t test. Each symbol represents an individual mouse (e) or independent culture (h, i). Data are representative of two independent experiments (a–i). Mean ± s.d. plotted in e, h, i. P values are listed in order from left to right. Source data are provided in the Source Data file.