Fig. 4: CUL5 inhibits IFN-β expression by suppressing mitochondrial antiviral-signaling protein (MAVS) O-GlcNAcylation in macrophages.

a Immunoblot of phosphorylated (P-)TBK1, TBK1, P-IRF3, IRF3, and β-actin in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. b Immunoblot of the indicated proteins in lysates of BMDMs stimulated by 4 μg/ml PolyU for the indicated times. c Confocal microscopic imaging of P-IRF3 distribution and expression in BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. Scale bars, 10 μm. d, e Dual-luciferase reporter system analysis of relative IFN-β-luc activation based on pRL-TK-luc with the indicated co-transfected plasmids. f Confocal microscopic imaging of BMDMs treated with 4 μg/ml Poly(I:C) for 4 h. Mitochondria, red; MAVS, green; nuclei stained with DAPI, blue. Yellow in merge indicates MAVS and mitochondria co-localization (white arrowheads). Scale bars, 10 μm. g Co-immunoprecipitation (Co-IP) and immunoblotting of HEK293T cells co-transfected with Flag-MAVS, HA-MAVS, and Myc-CUL5. h Gene ontology analysis of upregulated posttranslational modifications. X-axis: –log10 (p-value) calculated by Fisher’s exact test. i Immunoblot of total protein O-GlcNACylation in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. j Co-IP and immunoblotting of MAVS O-GlcNACylation in BMDMs treated with 4 μg/ml Poly(I:C) for 4 h. k Co-IP and immunoblotting of HEK293T cells co-transfected with HA-MAVS, Flag-OGT, and Myc-CUL5. l Immunoblotting of P-TBK1, TBK1, P-IRF3, IRF3, and β-actin in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times and pretreated with 20 μg/ml OSMI-1 for 30 min. m, n Dual-luciferase reporter system analysis of relative IFN-β-luc activation based on pRL-TK-luc with or without OSMI-1 treatment (m), or with the indicated co-transfected plasmids (n). o Heatmap summarizing Ifnb, Cxcl10, Isg15, and Ccl5 mRNA expression in BMDMs stimulated with 4 μg/ml Poly(I:C) for 6 h. Data in (a–c, f, g, i–l) are representative of three independent experiments. Data represent means ± s.e.m. of three biological replicates. p-values were calculated by two-way ANOVA (Sidak’s test) (d, e) or one-way ANOVA (Tukey’s test) (m, o). Source data are provided as a Source Data file.