murine ifn-β pbl Biolegend Search Results


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  • 99
    Millipore monoclonal anti beta actin antibody
    Monoclonal Anti Beta Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26462 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher puromycin
    Puromycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13754 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioLegend ifn β pbl
    Ifn β Pbl, supplied by BioLegend, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems recombinant ifn β
    SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with <t>IFN-β</t> (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p
    Recombinant Ifn β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    R&D Systems recombinant mouse ifn beta protein cf
    SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with <t>IFN-β</t> (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p
    Recombinant Mouse Ifn Beta Protein Cf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioLegend legend max mouse ifn β elisa kit
    SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with <t>IFN-β</t> (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p
    Legend Max Mouse Ifn β Elisa Kit, supplied by BioLegend, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    PBL Assay verikine human ifn β elisa kit
    SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with <t>IFN-β</t> (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p
    Verikine Human Ifn β Elisa Kit, supplied by PBL Assay, used in various techniques. Bioz Stars score: 96/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    R&D Systems recombinant ifn γ
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Recombinant Ifn γ, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PeproTech human ifn γ
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Human Ifn γ, supplied by PeproTech, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore β actin
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 49026 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sybr
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Sybr, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    BioLegend ccl5
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Ccl5, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega dual specific luciferase assay kit
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Dual Specific Luciferase Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    InvivoGen lipofectamine 2000
    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted <t>IFN-β</t> and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with <t>IFN-γ</t> (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P
    Lipofectamine 2000, supplied by InvivoGen, used in various techniques. Bioz Stars score: 97/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Sangon Biotech hsv120
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    Hsv120, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 89/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cycloheximide chx
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    Cycloheximide Chx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore nms 873
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    Nms 873, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen 3 cgamp
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    3 Cgamp, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems recombinant il 6
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    Recombinant Il 6, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 2000
    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and <t>HSV120</t> (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 357425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boster Bio cxcl10
    ZCCHC3 is essential for host defense against DNA virus in mice. a Effects of ZCCHC3-deficiency on serum levels of IFN-β, <t>CXCL10,</t> and IL-6 induced by DNA viruses. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 6–9 per strain, 8 weeks old) were infected with HSV-1, VACV or MCMV (3 × 10 7 , 5 × 10 6 , and 1 × 10 4 PFU per mouse respectively) for 6 h before measurement of the indicated serum cytokines by ELISA. Each symbol represents an individual mouse. b Measurement of viral genomic copy numbers and viral titers. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days. HSV-1 genomic copy numbers and viral titers in the brains of infected mice were quantified by qPCR and plaque assays respectively. c Effects of ZCCHC3-deficiency on HSV-1-or VACV-induced death of mice. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 7 per strain for HSV-1, n = 6 per strain for VACV, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 or VACV at 5 × 10 6 PFU per mouse, and the survival rates of mice were monitored daily. d Effects of ZCCHC3-deficiency on HSV-1-induced inflammation in the lungs of mice. Sex and age-matched Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days and lung sections were analyzed by H E staining. Scale bars, 100 μm. *P
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    Millipore polybrene
    ZCCHC3 is essential for host defense against DNA virus in mice. a Effects of ZCCHC3-deficiency on serum levels of IFN-β, <t>CXCL10,</t> and IL-6 induced by DNA viruses. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 6–9 per strain, 8 weeks old) were infected with HSV-1, VACV or MCMV (3 × 10 7 , 5 × 10 6 , and 1 × 10 4 PFU per mouse respectively) for 6 h before measurement of the indicated serum cytokines by ELISA. Each symbol represents an individual mouse. b Measurement of viral genomic copy numbers and viral titers. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days. HSV-1 genomic copy numbers and viral titers in the brains of infected mice were quantified by qPCR and plaque assays respectively. c Effects of ZCCHC3-deficiency on HSV-1-or VACV-induced death of mice. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 7 per strain for HSV-1, n = 6 per strain for VACV, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 or VACV at 5 × 10 6 PFU per mouse, and the survival rates of mice were monitored daily. d Effects of ZCCHC3-deficiency on HSV-1-induced inflammation in the lungs of mice. Sex and age-matched Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days and lung sections were analyzed by H E staining. Scale bars, 100 μm. *P
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    Solulink streptavidin agarose
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Streptavidin Agarose, supplied by Solulink, used in various techniques. Bioz Stars score: 93/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vps34 in1
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Vps34 In1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend tnfα
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    Tnfα, supplied by BioLegend, used in various techniques. Bioz Stars score: 97/100, based on 959 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore k48 linked polyubiquitin
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
    K48 Linked Polyubiquitin, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical type ii collagenase
    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and <t>streptavidin-Sepharose</t> beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P
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    Image Search Results


    SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p

    Journal: PLoS Pathogens

    Article Title: SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA

    doi: 10.1371/journal.ppat.1007336

    Figure Lengend Snippet: SNX8 is essential for DNA virus-triggered signaling. (A) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1, VV, or ECTV (MOI = 1) for 6 h before qPCR analysis. (B ) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (C) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for 18 h. The culture media were collected for ELISA. (D) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were infected with HSV-1 (MOI = 1) for the indicated times before immunoblot analysis. (E) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 3 h before qPCR analysis. (F) Snx8 +/+ and Snx8 -/- BMDMs (4x10 5 ) were transfected with the indicated nucleic acids (3 μg/ml) for 18 h. The culture media were collected for ELISA. (*p

    Article Snippet: Reagents, antibodies, cells and viruses 3′3′-cGAMP, Lipofectamine 2000 (InvivoGen); poly(dA:dT), DNA90, ISD, HSV60, HSV120 (Sangon Biotech); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin and VPS34-IN1 (Sigma); puromycin (Thermo); recombinant IFN-β (R & D Systems); ELISA kits for murine IFN-α and IFN-β (PBL) and IL-6 (Biolegend).

    Techniques: Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection

    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and HSV120 (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p

    Journal: PLoS Pathogens

    Article Title: SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA

    doi: 10.1371/journal.ppat.1007336

    Figure Lengend Snippet: SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and HSV120 (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p

    Article Snippet: Reagents, antibodies, cells and viruses 3′3′-cGAMP, Lipofectamine 2000 (InvivoGen); poly(dA:dT), DNA90, ISD, HSV60, HSV120 (Sangon Biotech); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin and VPS34-IN1 (Sigma); puromycin (Thermo); recombinant IFN-β (R & D Systems); ELISA kits for murine IFN-α and IFN-β (PBL) and IL-6 (Biolegend).

    Techniques: Transfection, Luciferase, Infection, Expressing, Real-time Polymerase Chain Reaction

    SNX8 is associated with MITA. (A) Snx8 +/+ and Snx8 -/- MLFs (4x10 5 ) were left un-treated or treated with cGAMP (0.2 μg/ml) for 3 h before qPCR analysis. (B) Snx8 +/+ and Snx8 -/- MLFs (4x10 5 ) were left un-treated or treated with cGAMP (0.2 μg/ml) for the indicated times before immunoblot analysis. (C) HEK293 cells (1x10 5 ) were first transfected with increased amounts of SNX8 RNAi (0.4 μg) for 24 h, and then re-transfected with the IFN-β reporter (0.05 μg) and indicated expression plasmids (0.1 μg each) for 24 h before luciferase assays. (*p

    Journal: PLoS Pathogens

    Article Title: SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA

    doi: 10.1371/journal.ppat.1007336

    Figure Lengend Snippet: SNX8 is associated with MITA. (A) Snx8 +/+ and Snx8 -/- MLFs (4x10 5 ) were left un-treated or treated with cGAMP (0.2 μg/ml) for 3 h before qPCR analysis. (B) Snx8 +/+ and Snx8 -/- MLFs (4x10 5 ) were left un-treated or treated with cGAMP (0.2 μg/ml) for the indicated times before immunoblot analysis. (C) HEK293 cells (1x10 5 ) were first transfected with increased amounts of SNX8 RNAi (0.4 μg) for 24 h, and then re-transfected with the IFN-β reporter (0.05 μg) and indicated expression plasmids (0.1 μg each) for 24 h before luciferase assays. (*p

    Article Snippet: Reagents, antibodies, cells and viruses 3′3′-cGAMP, Lipofectamine 2000 (InvivoGen); poly(dA:dT), DNA90, ISD, HSV60, HSV120 (Sangon Biotech); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin and VPS34-IN1 (Sigma); puromycin (Thermo); recombinant IFN-β (R & D Systems); ELISA kits for murine IFN-α and IFN-β (PBL) and IL-6 (Biolegend).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Expressing, Luciferase

    iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P

    Journal: PLoS Pathogens

    Article Title: iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA

    doi: 10.1371/journal.ppat.1006693

    Figure Lengend Snippet: iRhom2 is essential for SeV-triggered induction of downstream antiviral genes. (A) qPCR analysis of Ifnb1 and Il6 mRNAs in iRhom2 +/+ and iRhom2 ‒/‒ MEFs (left), BMDCs (middle) and BMDMs (right) infected with SeV for the indicated times. (B) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ cells un-infected or infected with SeV for 8 h (MEF) or the indicated times (BMDCs). (C) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– BMDMs un-infected or infected with SeV for the indicated times. (D) qPCR analysis of Gbp1 and Irf1 mRNAs in iRhom2 +/+ or iRhom2 ‒/‒ MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (E) Immunoblot analysis of the indicated proteins in iRhom2 +/+ and iRhom2 –/– MEFs untreated or treated with IFN-γ (100 ng/ml) for the indicated times. (F) qPCR analysis of Ifnb1 , Isg56 and Il6 mRNAs in iRhom2 +/+ and iRhom2 –/– MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. (G) ELISA of secreted IFN-β and IL-6 in iRhom2 +/+ or iRhom2 ‒/‒ MEFs transfected with poly (I:C) (3 μg/ml) for 6 h. * P

    Article Snippet: Reagents, antibodies, cells and viruses Poly(I:C) (InvivoGen); Recombinant IFN-γ (R & D Systems); cycloheximide (CHX), MG132, DBeQ, NMS-873 (Sigma-Aldrich); GM-CSF (peproTech); lipofectamine 2000 (Invitrogen); polybrene (Millipore); SYBR (BIO-RAD); dual-specific luciferase assay kit (Promega); ELISA kit for murine IFN-α and IFN-β (PBL); ELISA kits for murine TNF, IL-6 and CCL5 (Biolegend) were purchased from the indicated manufacturers.

    Techniques: Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Transfection

    Identification of iRhom2 as a positive regulator of SeV virus-triggered signaling. (A) Luciferase reporter assays with HEK293 cells that were co-transfected with the indicated reporter plasmids plus iRhom2 or empty vector (EV) and then left untreated (Mock) or treated with SeV or IFN-γ (100 ng/ml) for 12 h. (B) qPCR analysis of IFNB1 , ISG56 , IL6 , GBP1 and IRF1 mRNAs in HEK293 cells that were transfected with iRhom2 or empty vector (EV) and then treated with SeV for 8 h or IFN-γ (100 ng/ml) for 6 h. (C) qPCR analysis of IFNB1 , ISG56 , IP10 , IRF1 and IRHOM2 mRNAs in HEK293 cells that were transfected with control shRNA (Ctrl) and iRhom2-shRNA plasmid and then selected with puromycin (1 μg/ml) for 2 days before treatment with SeV for 8 h or IFN-γ (100 ng/ml) for 6 h. (D) Immunoblot analysis of the indicated proteins with HEK293 cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then infected with SeV for the indicated times. (E) Native gel analysis (above) and SDS-PAGE (below) of IRF3 dimerization with HEK293T cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then infected with SeV for the indicated times. (F)Immunoblot analysis of the indicated proteins with HEK293T cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then treated with IFN-γ (100 ng/ml) for the indicated times. *p

    Journal: PLoS Pathogens

    Article Title: iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA

    doi: 10.1371/journal.ppat.1006693

    Figure Lengend Snippet: Identification of iRhom2 as a positive regulator of SeV virus-triggered signaling. (A) Luciferase reporter assays with HEK293 cells that were co-transfected with the indicated reporter plasmids plus iRhom2 or empty vector (EV) and then left untreated (Mock) or treated with SeV or IFN-γ (100 ng/ml) for 12 h. (B) qPCR analysis of IFNB1 , ISG56 , IL6 , GBP1 and IRF1 mRNAs in HEK293 cells that were transfected with iRhom2 or empty vector (EV) and then treated with SeV for 8 h or IFN-γ (100 ng/ml) for 6 h. (C) qPCR analysis of IFNB1 , ISG56 , IP10 , IRF1 and IRHOM2 mRNAs in HEK293 cells that were transfected with control shRNA (Ctrl) and iRhom2-shRNA plasmid and then selected with puromycin (1 μg/ml) for 2 days before treatment with SeV for 8 h or IFN-γ (100 ng/ml) for 6 h. (D) Immunoblot analysis of the indicated proteins with HEK293 cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then infected with SeV for the indicated times. (E) Native gel analysis (above) and SDS-PAGE (below) of IRF3 dimerization with HEK293T cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then infected with SeV for the indicated times. (F)Immunoblot analysis of the indicated proteins with HEK293T cells that were transfected with iRhom2-shRNA plasmid and selected with puromycin (1 μg/ml) for 2 days and then treated with IFN-γ (100 ng/ml) for the indicated times. *p

    Article Snippet: Reagents, antibodies, cells and viruses Poly(I:C) (InvivoGen); Recombinant IFN-γ (R & D Systems); cycloheximide (CHX), MG132, DBeQ, NMS-873 (Sigma-Aldrich); GM-CSF (peproTech); lipofectamine 2000 (Invitrogen); polybrene (Millipore); SYBR (BIO-RAD); dual-specific luciferase assay kit (Promega); ELISA kit for murine IFN-α and IFN-β (PBL); ELISA kits for murine TNF, IL-6 and CCL5 (Biolegend) were purchased from the indicated manufacturers.

    Techniques: Luciferase, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, shRNA, Infection, SDS Page

    SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and HSV120 (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p

    Journal: PLoS Pathogens

    Article Title: SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA

    doi: 10.1371/journal.ppat.1007336

    Figure Lengend Snippet: SNX8 positively regulates DNA virus-triggered signaling. (A) HeLa cells (1x10 5 ) were transfected with IFN-β (0.05 μg), ISRE (0.05 μg) and NF-κB (5 ng) reporter plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg) for 24 h before luciferase assays were performed. (B) HeLa cells (1x10 5 ) were transfected with the IFN-β reporter (0.05 μg) plasmids and increased amounts of SNX8 (0.05, 0.1, 0.2 μg). Twenty hours after transfection, cells were left un-infected or infected with HSV-1 (MOI = 1) for 10 h before luciferase assays were performed. (C) SNX8 stable-expressing HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD and HSV120 (3 μg/ml) for 6 h before qPCR analysis. (D) SNX8-deficient HFFs (4x10 5 ) were infected with HSV-1 (MOI = 1) or transfected with ISD or HSV120 (3 μg/ml) for 6 h before qPCR analysis. (E) SNX8-deficient HFFs (4x10 5 ) were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. (F) SNX8-deficient HFFs (4x10 5 ) were left un-infected or infected with HSV-1 (MOI = 1) for the indicated times followed by immunoblot analysis. (*p

    Article Snippet: 3′3′-cGAMP, Lipofectamine 2000 (InvivoGen); poly(dA:dT), DNA90, ISD, HSV60, HSV120 (Sangon Biotech); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin and VPS34-IN1 (Sigma); puromycin (Thermo); recombinant IFN-β (R & D Systems); ELISA kits for murine IFN-α and IFN-β (PBL) and IL-6 (Biolegend).

    Techniques: Transfection, Luciferase, Infection, Expressing, Real-time Polymerase Chain Reaction

    ZCCHC3 is essential for host defense against DNA virus in mice. a Effects of ZCCHC3-deficiency on serum levels of IFN-β, CXCL10, and IL-6 induced by DNA viruses. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 6–9 per strain, 8 weeks old) were infected with HSV-1, VACV or MCMV (3 × 10 7 , 5 × 10 6 , and 1 × 10 4 PFU per mouse respectively) for 6 h before measurement of the indicated serum cytokines by ELISA. Each symbol represents an individual mouse. b Measurement of viral genomic copy numbers and viral titers. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days. HSV-1 genomic copy numbers and viral titers in the brains of infected mice were quantified by qPCR and plaque assays respectively. c Effects of ZCCHC3-deficiency on HSV-1-or VACV-induced death of mice. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 7 per strain for HSV-1, n = 6 per strain for VACV, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 or VACV at 5 × 10 6 PFU per mouse, and the survival rates of mice were monitored daily. d Effects of ZCCHC3-deficiency on HSV-1-induced inflammation in the lungs of mice. Sex and age-matched Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days and lung sections were analyzed by H E staining. Scale bars, 100 μm. *P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 is essential for host defense against DNA virus in mice. a Effects of ZCCHC3-deficiency on serum levels of IFN-β, CXCL10, and IL-6 induced by DNA viruses. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 6–9 per strain, 8 weeks old) were infected with HSV-1, VACV or MCMV (3 × 10 7 , 5 × 10 6 , and 1 × 10 4 PFU per mouse respectively) for 6 h before measurement of the indicated serum cytokines by ELISA. Each symbol represents an individual mouse. b Measurement of viral genomic copy numbers and viral titers. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days. HSV-1 genomic copy numbers and viral titers in the brains of infected mice were quantified by qPCR and plaque assays respectively. c Effects of ZCCHC3-deficiency on HSV-1-or VACV-induced death of mice. Zcchc3 +/+ and Zcchc3 − / − mice ( n = 7 per strain for HSV-1, n = 6 per strain for VACV, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 or VACV at 5 × 10 6 PFU per mouse, and the survival rates of mice were monitored daily. d Effects of ZCCHC3-deficiency on HSV-1-induced inflammation in the lungs of mice. Sex and age-matched Zcchc3 +/+ and Zcchc3 − / − mice ( n = 3 per strain, 8 weeks old) were intra-nasally infected with HSV-1 at 3 × 10 7 PFU per mouse for 6–7 days and lung sections were analyzed by H E staining. Scale bars, 100 μm. *P

    Article Snippet: Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Staining

    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Article Snippet: Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Article Snippet: Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Microscale Thermophoresis, Recombinant, Purification, Staining, Infection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing

    ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P

    Article Snippet: Reagents, antibodies, cells, and viruses Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

    ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Journal: Nature Communications

    Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

    doi: 10.1038/s41467-018-05559-w

    Figure Lengend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P

    Article Snippet: Reagents, antibodies, cells, and viruses Poly(dA:dT), 2′ 3′-cGAMP, and Lipofectamine 2000 (InvivoGen); polybrene (Millipore); SYBR (Bio-Rad); FuGene and Dual-Specific Luciferase Assay Kit (Promega); digitonin (Sigma); puromycin and EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); ELISA kits for murine IFN-β (PBL), IL-6 (BioLegend), and CXCL10 (BOSTER); recombinant IFN-β (R & D systems) were purchased for the indicated manufacturers.

    Techniques: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Microscale Thermophoresis, Recombinant, Purification, Staining, Infection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing