Journal: Nature communications

Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination.

doi: 10.1038/s41467-019-12145-1

Figure Lengend Snippet: Fig. 4 USP19 acts at the level of TRIF. a Overexpression of USP19 inhibits TRIF-mediated ISRE activation. HEK293 cells were transfected with ISRE reporter and the indicated adapter expression plasmids together with a control or USP19 expression plasmid for 24 h before luciferase assays were performed. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01 (unpaired t-test); N.S., not significant. b USP19 interacts with TRIF in overexpression systems. HEK293 cells were transfected with HA-USP19 and the indicated adapter expression plasmids. Twenty hours after transfection, co- immunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with anti-HA or anti-Flag. c Domain mapping of the interaction between USP19 and TRIF. HEK293 cells were transfected with the indicated truncations before co- immunoprecipitation and immunoblotting analysis with the indicated antibodies. The schematic presentations of USP19 and TRIF truncations are shown at the top. d Effects of USP19 and its truncations on poly(I:C)- and LPS-induced IFN-β promoter activation. The 293-TLR3 or 293-TLR4 cells were transfected with the IFN-β promoter reporter and Flag-USP19 or its truncations for 24 h, and then left untreated, treated with poly(I:C) (50 μg/mL) or LPS (100 ng/ml) respectively for 8 h before luciferase assays. Graphs show mean ± SD; n = 3 independent samples. **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Dunnett’s test); N.S., not significant. e Endogenous USP19 is associated with TRIF. The 293-TLR3 or 293-TLR4 cells were left untreated or treated with poly (I:C) (100 μg/ml) or LPS (100 ng/ml) respectively for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file. Error bars represent standard deviation of the mean

Article Snippet: Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and D-galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies.

Techniques: Over Expression, Activation Assay, Transfection, Expressing, Control, Plasmid Preparation, Luciferase, Immunoprecipitation, Western Blot, Standard Deviation

Journal: Nature communications

Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination.

doi: 10.1038/s41467-019-12145-1

Figure Lengend Snippet: Fig. 6 USP19-mediated deubiquitination of TRIF impairs its function. a Overexpression of USP19 inhibits the interaction of TRIF with TLR3 but not with TBK1 or TRAF3. HEK293 cells were transfected with Flag-TLR3, Flag-TBK1, or Flag-TRAF3 and HA-TRIF together with a control or Myc-USP19 expression plasmid for 20 h. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates and lysates were analyzed by immunoblotting with anti-HA or anti-Flag. b Overexpression of USP19 but not USP19(C607S) inhibits TRIF-TLR3 interaction. HEK293 cells were transfected with Flag-TLR3 or Flag-TBK1 and HA-TRIF together with a control or Myc-USP19 expression plasmid for 20 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c USP19-deficiency potentiates TLR3-TRIF association. The control or USP19-deficient 293- TLR3 cells were treated with poly(I:C) (100 μg/ml) or left untreated for the indicated times. Cell lysates were immunoprecipitated with anti-TRIF or control IgG. The immunoprecipitates were analyzed by immunoblots with the indicated antibodies. d Overexpression of USP19 but not USP19(C607S) inhibits TRIF-TLR4 and TRIF-TRAM interactions. HEK293 cells were transfected with the indicated plasmids for 20 h before coimmunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. e USP19-deficiency increases TLR4-TRIF association. The control or USP19- deficient 293-TLR4 cells were treated with LPS (100 ng/ml) or left untreated for the indicated times before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file

Article Snippet: Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and D-galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation