murine ifn-β pbl Biolegend Search Results


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  • 99
    Thermo Fisher puromycin
    Puromycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 33074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioLegend ifn β pbl
    Ifn β Pbl, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    R&D Systems recombinant ifn β
    ZCCHC3 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the <t>IFN-β</t> promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis. * P
    Recombinant Ifn β, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant mouse ifn beta protein cf
    ZCCHC3 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the <t>IFN-β</t> promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis. * P
    Recombinant Mouse Ifn Beta Protein Cf, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Assay human ifn β
    C-di-AMP induces <t>IFN-β</t> production in murine dendritic cells (DCs). ( A ) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo . Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm 2 /steridian). Error bars are SEM. ( B ) C-di-AMP induces IFN-β production in DCs in vitro . BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.
    Human Ifn β, supplied by PBL Assay, used in various techniques. Bioz Stars score: 91/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend legend max mouse ifn β elisa kit
    C-di-AMP induces <t>IFN-β</t> production in murine dendritic cells (DCs). ( A ) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo . Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm 2 /steridian). Error bars are SEM. ( B ) C-di-AMP induces IFN-β production in DCs in vitro . BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.
    Legend Max Mouse Ifn β Elisa Kit, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Assay verikine human ifn β elisa kit
    C-di-AMP induces <t>IFN-β</t> production in murine dendritic cells (DCs). ( A ) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo . Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm 2 /steridian). Error bars are SEM. ( B ) C-di-AMP induces IFN-β production in DCs in vitro . BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.
    Verikine Human Ifn β Elisa Kit, supplied by PBL Assay, used in various techniques. Bioz Stars score: 88/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 67284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Assay verikine mouse interferon alpha interferon beta elisa kits
    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
    Verikine Mouse Interferon Alpha Interferon Beta Elisa Kits, supplied by PBL Assay, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PBL Assay verikine human interferon alpha interferon beta elisa 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
    Verikine Human Interferon Alpha Interferon Beta Elisa Kit, supplied by PBL Assay, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    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: 93/100, based on 1124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mg132
    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
    Mg132, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dbeq
    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Promega fugene
    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with <t>MG132</t> for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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    ZCCHC3 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the IFN-β promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting 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 positively regulates dsDNA-triggered signaling. a ZCCHC3 activates the IFN-β promoter in a dose-dependent manner. HEK293 cells were transfected with the IFN-β reporter and increased amounts of ZCCHC3 plasmid for 18 h, and then left un-infected or infected with HSV-1 for 12 h before luciferase assays. b Effects of ZCCHC3 on transcription of downstream genes induced by HSV-1. Control HFF cells and HFFs stably expressing ZCCHC3 were infected with HSV-1 for the indicated times before qPCR analysis. c Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. Control and HFFs stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. d Effects of ZCCHC3-deficiency on transcription of downstream genes induced by HSV-1. ZCCHC3- KO HFFs were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before qPCR analysis. ZCCHC3-deficiency in the KO cells was confirmed by immunoblotting analysis (right blots). e Effects of ZCCHC3 on transcription of downstream genes induced by cytosolic dsDNA. ZCCHC3-KO and control HFFs were transfected with the indicated nucleic acids (3 μg/ml) for 4 h before qPCR analysis. f Effects of ZCCHC3-deficiency on HSV-1-induced phosphorylation of TBK1 and IRF3. ZCCHC3-KO and control HFFs were left un-infected or infected with HSV-1 for the indicated times before immunoblotting analysis. g Effects of ZCCHC3-deficiency on IFN-β-induced transcription of downstream genes. Control and ZCCHC3- KO THP1 cells were generated by the CRISPR-Cas9 method. The cells were left un-treated or treated with IFN-β (100 ng/ml) for 6 h before qPCR analysis. h Effects of ZCCHC3-deficiency on IFN-β-induced phosphorylation of STAT1. ZCCHC3-KO and control THP1 cells were left un-treated or treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting 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: Transfection, Plasmid Preparation, Infection, Luciferase, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Generated, CRISPR

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

    C-di-AMP induces IFN-β production in murine dendritic cells (DCs). ( A ) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo . Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm 2 /steridian). Error bars are SEM. ( B ) C-di-AMP induces IFN-β production in DCs in vitro . BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.

    Journal: PLoS ONE

    Article Title: The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages

    doi: 10.1371/journal.pone.0095728

    Figure Lengend Snippet: C-di-AMP induces IFN-β production in murine dendritic cells (DCs). ( A ) C-di-AMP targets the IFNB promoter in DCs and monocytes/macrophages/granulocytes in vivo . Mice of indicated phenotypes were i. n. treated with c-di-AMP: “CD4+” indicates T cell specific, “CD19+” B cell specifc, “LysM+” monocyte/macrophage/granulocyte specific, and “CD11c+” DC specific control of luciferase expression by the IFN-β promoter. Quantification of in vivo imaging signals derived from luciferase activity at different time points is shown for n = 5. Results are expressed as average of radiance (in photons/s/cm 2 /steridian). Error bars are SEM. ( B ) C-di-AMP induces IFN-β production in DCs in vitro . BMDCs were cultured in the presence of GM-CSF or Flt3l, as indicated on the x axis. IFN-β secretion was determined in the culture medium by ELISA. Error bars are SEM, n = 3.

    Article Snippet: IFN-β detection in cell culture medium Murine IFN-β was analyzed using the Legend Max Mouse IFN-β ELISA kit with pre-coated plates (BioLegend, San Diego, California, USA); human IFN-β was analyzed using the VeriKine human IFN-β ELISA kit (PBL Interferon Source, Piscataway, New Jersey, USA).

    Techniques: In Vivo, Mouse Assay, Luciferase, Expressing, In Vivo Imaging, Derivative Assay, Activity Assay, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

    C-di-AMP preferentially activates human myeloid dendritic cells (DCs) in vitro . PBMC-derived human plasmacytoid DCs (pDC) or myeloid (conventional) DCs (mDC) were incubated for 24 h in the presence of 60 µg/ml c-di-AMP or without additive (control). ( A ) Cells were decorated with fluorochrome-conjugated antibodies specific for the identification markers CD11c (mDC), CD303 (pDC) CD80, CD83 and CD86, and analyzed by flow cytometry. Normalized median fluorescence intensity (MDFI) is shown as fold increase compared to the control. Error bars show SEM for n = 3. ( B ) The medium from the cultured mDC or pDC was analyzed for IFN-β secretion by ELISA. Error bars show SEM for n = 3. Differences are statistically significant at p

    Journal: PLoS ONE

    Article Title: The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages

    doi: 10.1371/journal.pone.0095728

    Figure Lengend Snippet: C-di-AMP preferentially activates human myeloid dendritic cells (DCs) in vitro . PBMC-derived human plasmacytoid DCs (pDC) or myeloid (conventional) DCs (mDC) were incubated for 24 h in the presence of 60 µg/ml c-di-AMP or without additive (control). ( A ) Cells were decorated with fluorochrome-conjugated antibodies specific for the identification markers CD11c (mDC), CD303 (pDC) CD80, CD83 and CD86, and analyzed by flow cytometry. Normalized median fluorescence intensity (MDFI) is shown as fold increase compared to the control. Error bars show SEM for n = 3. ( B ) The medium from the cultured mDC or pDC was analyzed for IFN-β secretion by ELISA. Error bars show SEM for n = 3. Differences are statistically significant at p

    Article Snippet: IFN-β detection in cell culture medium Murine IFN-β was analyzed using the Legend Max Mouse IFN-β ELISA kit with pre-coated plates (BioLegend, San Diego, California, USA); human IFN-β was analyzed using the VeriKine human IFN-β ELISA kit (PBL Interferon Source, Piscataway, New Jersey, USA).

    Techniques: In Vitro, Derivative Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay

    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: 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

    Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with MG132 for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

    Journal: The Journal of Experimental Medicine

    Article Title: Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5

    doi: 10.1084/jem.20161015

    Figure Lengend Snippet: Physiological relationship of sumoylation and K48-linked polyubiquitination of MDA5. (A) Identification of the residue in MDA5-CARD targeted by RNF125 for K48-linked polyubiquitination. HEK293 cells were transfected with the indicated plasmids for 18 h, and then treated with MG132 for 6 h before immunoprecipitation and immunoblotting analysis. (B) Dynamic sumoylation and K48-linked polyubiquitination of MDA5 and its mutants after EMCV stimulation in reconstituted cells. MDA5-shRNA MEFs reconstituted with MDA5 or its mutants were infected with EMCV for the indicated times, followed by immunoprecipitation and immunoblotting analysis (top histogram). As described in Fig. 4 G , superinfection (K43R-H) or subinfection (K128R-L) of the MDA5 mutant-containing retroviruses was used to make the reconstituted MDA5 and its mutants express at similar levels. (C) The schematic diagram for dynamic K48-linked polyubiquitination and sumoylation of MDA5 before and after viral infection. The crystal structure of MDA5 Helicase domain (PDB: 4GL2) was obtained from the PDB database. Black, ubiquitination sites; red, sumoylation sites; blue, phosphorylation site. (D) Effects of simultaneous mutation of K128 and K865 on EMCV-induced K48-linked polyubiquitination of MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h, and then cells were left uninfected or infected with EMCV for 12 h, followed by immunoprecipitation and immunoblotting analysis. (E) EMCV-induced transcription of downstream antiviral genes in MDA5-shRNA MEFs reconstituted with wild-type MDA5 or its mutants. The reconstituted cells were left uninfected or infected with EMCV for 6 h before qPCR analysis. Data in E are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

    Article Snippet: Reagents, antibodies, viruses, and cells The following reagents were used: GM-CSF (PeproTech); poly(I:C) and poly(I:C)-LMW (InvivoGen); cycloheximide (CHX), MG132, N-ethylmaleimide (NEM; Sigma-Aldrich); Lipofectamine 2000 (Invitrogen); polybrene (EMD Millipore); SYBR (Bio-Rad laboratories); RNase inhibitor (Thermo Fisher Scientific); ELISA kit for murine Ifn-β (PBL); ELISA kit for murine Tnfα (BioLegend); mouse monoclonal antibodies against HA (Covance); Flag and β-actin (Sigma-Aldrich); phospho-IκBα (S536; Cell Signaling Technology); rabbit polyclonal antibodies against phospho-IRF3(S396; Cell Signaling Technology), phospho-TBK1(S172; Abcam), SUMO1 (Abclone Biotechnology), K63-lined polyubiquitin and K48-linked polyubiquitin (EMD Millipore) were purchased from the indicated manufacturers.

    Techniques: Transfection, Immunoprecipitation, shRNA, Infection, Mutagenesis, Real-time Polymerase Chain Reaction