Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B is critical for IFN-I induction by and control of HSV-1 infection in vivo. a Immunoblots showing Ubxn3b knockout efficiency in various tissues without/with tamoxifen (TMX) treatment in Cre +/− Ubxn3b flox/flox mice. Tubulin is a housekeeping protein control. b , c The survival curves of tamoxifen (TMX)-treated Ubxn3b flox/flox , TMX-treated Cre +/− , mock-treated Cre +/− Ubxn3b flox/flox (designated Ubxn3b +/+ ) and TMX-treated Cre +/− Ubxn3b flox/flox (designated Ubxn3b −/− ) littermate and Sting −/− mice challenged with 1 × 10 7 plaque-forming units (PFU) per mouse of HSV-1 i.v. N = 8–9 mice/genotype. *** P < 0.001 (log-rank test). d The serum IFN-I concentrations (mean ± s.e.m) of mice challenged with 2 × 10 6 PFU per mouse of HSV-1 i.v. ** P < 0.01; * P < 0.05 (non-parametric Mann–Whitney test), N = 10 ( Ubxn3b +/+ ) or N = 6 ( Ubxn3b −/− ). e The viral titers (mean ± s.e.m) in the brain on day 3 after infection (PFU per gram tissue). ** P < 0.01 (non-parametric Mann–Whitney test), N = 5 mice per genotype. The data are pooled from two independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Infection, In Vivo, Western Blot, Knock-Out, MANN-WHITNEY

Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B is critical for STING-dependent IFN-I induction in mouse primary cells. a ELISA of IFN-α in the cell culture supernatants of ( Ubxn3b +/+ , Ubxn3b −/− ) bone marrow-derived macrophages (M ϕ ), Flt3-induced pDCs, and GM-CSF-induced cDCs (pooled from five littermates) 20 h after the indicated treatments. N = 3 per genotype. * P < 0.05 (unpaired Student’s t test). b Immunoblots of an interferon-stimulated gene Oas1a, Sting, and Ubxn3b expression in cDCs 20 h after the indicated treatments. Tubulin is a housekeeping protein control. qPCR analysis of ( c ) Ifnb1 and Tnfa mRNA expression and d cellular HSV-1 genome loads in cDCs infected with HSV-1 (MOI = 10) for the indicated time. e Immunoblots showing Ubxn3b and housekeeping Gapdh protein expression in mock ( Ubxn3b +/+ ) or 4-hydroxyl tamoxifen-treated Cre +/− Ubxn3b flox/flox ( Ubxn3b −/− ) MEFs. f Viral titers (plaque-forming units/ml) in the supernatant of MEFs infected with HSV-1 (MOI = 0.1). N = 3 per genotype per time point. * P < 0.05; ** P < 0.01 (unpaired Student’s t test). g qPCR analysis of selected immune gene mRNA expression in MEFs infected with HSV-1 as in g . h The immunoblots show knockout efficacy of STING and UBXN3B by CRISPR-Cas9 in human primary trophoblasts. Actin is a housekeeping protein control. i Fluorescent microscopic images of human primary trophoblasts infected with HSV-1-GFP (MOI = 0.3) for 18 h. Objective, ×5. Scale bar, 10 µm. j qPCR analysis of Ifnb1 mRNA expression in human primary trophoblasts infected with HSV-1-GFP for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR ( N = 2 per genotype per time point). * P < 0.05; ** P < 0.01 (unpaired Student’s t test). The results are representative of two independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Derivative Assay, Western Blot, Expressing, Control, Infection, Knock-Out, CRISPR

Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B is critical for type I IFN responses to SeV and VSV, but not EMCV infection. a The survival curves of Ubx3b −/− ( N = 6) and Ubxn3b −/− ( N = 7) mice infected with 1 × 10 7 PFU of VSV i.v. The results are pooled from two experiments. * P < 0.05 (log-rank test). b , c qPCR analysis of Ifnb1 expression in cDCs infected with b VSV (MOI = 5) or c SeV (200 hemagglutination units per 10 5 cells). d ELISA of IFN-α in the cell culture supernatants of cDCs infected with SeV and VSV as in b , c . N = 3 per genotype per time point. * P < 0.05 (unpaired Student’s t test). e The survival curves of Ubx3b +/+ ( N = 7) and Ubxn3b −/− ( N = 8) mice infected with 200 PFU of EMCV i.p. P = 0.53 (log-rank test). The results are pooled from two experiments. f qPCR analysis of Ifnb1 expression in cDCs infected with EMCV (MOI = 5) for the indicated time. Bars/data points: mean ± s.e.m. Two biological replicates were pooled for qPCR ( N = 2 per genotype per time point). * P < 0.05 (unpaired Student’s t test). The results are representative of two independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B regulates STING dimerization, phosphorylation, and degradation. a Immunoblots showing Sting dimerization in untreated ( Ubxn3b +/+ ) and 4-hydroxyl tamoxifen-induced Cre +/− Ubxn3b flox/flox ( Ubxn3b −/− ) primary MEFs. The cells were infected without (mock) or with HSV-1 (MOI = 0.5) for 8 h. b Immunoblotting analysis of the whole-cell lysates of bone marrow-derived cDCs infected with HSV-1 (MOI = 5). Mono monomer, 2-ME β-mercaptoethanol, a chemical compound that reduces disulfide bonds. c qPCR quantification of Ifnb1 and Tnfa mRNA induction in cDCs by HSV-1 (MOI = 5). Bars: mean ± s.e.m. Two biological replicates were pooled for qPCR ( N = 2 per genotype per time point). * P < 0.05 (unpaired Student’s t test). d – f Immunoblotting analysis of the whole-cell lysates of d MEFs infected with HSV-1 (MOI = 0.5), e MEFs transfected with ISD (8 µg/ml) in the absence or presence of 40 µM of chloroquine (+CQ) and f trophoblasts transfected with cGAMP (8 µg/ml). In e , f , the arrows indicate phosphorylated STING with long and short exposure. Actin, Tubulin (Tub), and GAPDH are housekeeping protein controls. g qPCR quantification of IFNB1 and TNFA mRNA induction in trophoblasts transfected with cGAMP (8 µg/ml). In c , g , the bars are: mean ± s.e.m. Two biological replicates were pooled for qPCR ( N = 2 per genotype per time point). * P < 0.05 (unpaired Student’s t test). The results are representative of two to three independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Infection, Derivative Assay, Transfection

Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B interacts with STING and TRIM56. a Co-immunoprecipitation (co-IP) of STING with UBXN3B from HEK293 cells transfected with FLAG-UBXNs and HA-STING plasmids using anti-FLAG magnetic beads, followed by immunoblotting (IB) with an anti-HA (STING) and FLAG (UBXN) antibody. b Immunofluorescence staining of STING and UBXN3B in H1975 cells treated without (Mock) or with ISD (8 µg/ml) for 3 h. The cells were stained with a mouse anti-human STING and rabbit anti-UBXN3B antibody followed by a secondary antibody conjugated with Alexa Fluor 594/488. The nuclei were stained with DAPI. Objective, ×40. Scale bar, 200 µm. c A schematic diagram of UBXN3B functional domains. d Co-IP of STING with the truncated forms of UBXN3B. The procedures are similar to b . e Co-IP of UBXN3B with TRIM from HEK293 cells transfected with FLAG-UBXN3B and Myc-TRIM plasmids using anti-Myc magnetic beads, followed by IB with an anti-Myc (TRIM) and FLAG (UBXN3B) antibody. f Co-IP of TRIM56 with the truncated forms of UBXN3B from HEK293 cells transfected with FLAG-UBXN3B truncates and Myc-TRIM56 plasmids using anti-FLAG magnetic beads, followed by IB with an anti-Myc (TRIM56) and FLAG (UBXN truncates) antibody. GAPDH is a housekeeping protein control. WCE whole-cell extract. The results are representative of two independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Magnetic Beads, Western Blot, Immunofluorescence, Staining, Functional Assay, Control

Journal: Nature Communications

Article Title: UBXN3B positively regulates STING-mediated antiviral immune responses

doi: 10.1038/s41467-018-04759-8

Figure Lengend Snippet: UBXN3B mediates STING interaction with and ubiquitination by TRIM56. a WT and Sting gt/gt MEFs were infected with or without HSV-1 (MOI = 0.5) for 8 h. Sting was immunoprecipitated (IP) from the MEF lysates using a rabbit anti-Sting antibody. The immunoblots indicate Sting co-immunoprecipitation with Ubxn3b and Trim56 after viral infection. WCE whole-cell extract. b WT and Ubxn3b −/− MEFs were transfected with vector or FLAG-UBXN3B plasmids by electroporation, and then infected with HSV-1 (MOI = 0.5) for 8 h. Sting IP was performed as in a . Rabbit IgG was used as a negative control. c The immunoblots show knockout efficacy of UBXN3B in HEK293T-STING cell line. d WT and UBXN3B −/− cells (parent cell line is HEK293T-FLAG-STING) were transfected with the indicated combinations of plasmids. FLAG-STING was immunoprecipitated (IP) with anti-FLAG magnetic beads. The proteins in IP and WCE were immunoblotted by an anti-K63-linked polyubiquitin, anti-Myc (UBXN3B and TRIM56) and anti-FLAG (STING) antibody, respectively. e Sting was precipitated with a rabbit anti-Sting antibody from cDCs infected without or with HSV-1 (MOI = 10) for 4 and 8 h. The Sting −/− cDCs were used as an IP control. The proteins in IP and WCE were immunoblotted with an anti-K63 and Sting antibody, respectively. Tubulin and GAPDH are housekeeping controls. The results are representative of two independent experiments

Article Snippet: The rabbit anti-GAPDH (D16H11, Cat# 5174, 1:3000), calreticulin (D3E6, Cat# 12238, 1:200 for IFA), α-Tubulin (Cat# 2144, 1:3000), UBXN3B (D8H6D, Cat# 34945, 1:1000 for IB, 1:100 for IFA), TBK1 (D1B4, Cat# 3504, 1:1000), phospho-TBK1 (D52C2, Cat# 5483, 1:500), K63-linked polyubiquitin (D7A11, Cat# 5621,1:500), phospho-IRF3 (4D4G, Cat# 4947, 1:500), human phospho-STING (Cat# 85735, 1:500), and STING (D2P2F, Cat#13647, 1:500) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Infection, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Electroporation, Negative Control, Knock-Out, Magnetic Beads, Control