ez link psoralen peg3 biotin Search Results


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  • 99
    Qiagen qiaquick gel extraction kit
    Qiaquick Gel Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 81812 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polybrene
    Polybrene, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 52866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ez link psoralen peg3 biotin
    Ez Link Psoralen Peg3 Biotin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    InvivoGen lipofectamine 2000
    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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    92
    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
    Cxcl10, supplied by Boster Bio, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sybr
    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
    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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    93
    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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    Promega dual specific luciferase assay kit
    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
    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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    InvivoGen poly i c lwm
    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
    Poly I C Lwm, supplied by InvivoGen, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    InvivoGen poly i c fluorescein
    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
    Poly I C Fluorescein, supplied by InvivoGen, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dtt
    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
    Dtt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech 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 PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lysosome isolation kit
    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
    Lysosome Isolation Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ht dna
    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
    Ht Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin agarose resin
    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
    Streptavidin Agarose Resin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare protein g sepharose
    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
    Protein G Sepharose, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 11955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and <t>Iκbα.</t> HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P
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    <t>G3BP1</t> targets at upstream of MAVS. a , b Effects of G3BP1 knockdown on the IFN-β promoter and ISRE activation. The control or stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter a or ISRE reporter b (0.1 μg), TK (0.02 μg), and the indicated plasmids (0.1 μg each) for 24 h followed by luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P
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    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of <t>IRF3</t> and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
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    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of <t>IRF3</t> and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
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    Image Search Results


    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

    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: 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: 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

    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Journal: Cell Research

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Transfection, Infection, Luciferase, Over Expression, Stable Transfection, Software, Plasmid Preparation

    Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Infection, Luciferase, Software

    G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Transfection, Luciferase, Infection, Software

    Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P

    Journal: Cell Research

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activation Assay

    G3BP1 interacts with RIG-I. a G3BP1 interacts with RIG-I but not with MDA5, MAVS, TBK1, or IRF3 in the overexpression system. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg of each) for 24 h. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RIG-I in HEK293T cells. HEK293T cells (5 × 10 7 ) were uninfected or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Domain identification of G3BP1 and RIG-I interaction. HEK293T cells were transfected with the expression plasmids encoding RIG-I and G3BP1 or the corresponding mutants (5 μg each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. e , f Effects of G3BP1 overexpression and its mutants on SeV-triggered IFN-β promoter and ISRE activation. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter (0.1 μg) and the indicated expression plasmids (0.1 μg) for 24 h. Then cells were uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV empty vector, Luc luciferase, αF anti-Flag, αH anti-HA, HC heavy chain, WT wild-type.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 interacts with RIG-I. a G3BP1 interacts with RIG-I but not with MDA5, MAVS, TBK1, or IRF3 in the overexpression system. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg of each) for 24 h. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RIG-I in HEK293T cells. HEK293T cells (5 × 10 7 ) were uninfected or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Domain identification of G3BP1 and RIG-I interaction. HEK293T cells were transfected with the expression plasmids encoding RIG-I and G3BP1 or the corresponding mutants (5 μg each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. e , f Effects of G3BP1 overexpression and its mutants on SeV-triggered IFN-β promoter and ISRE activation. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter (0.1 μg) and the indicated expression plasmids (0.1 μg) for 24 h. Then cells were uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV empty vector, Luc luciferase, αF anti-Flag, αH anti-HA, HC heavy chain, WT wild-type.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Infection, Expressing, Activation Assay, Luciferase, Immunoprecipitation, Plasmid Preparation

    G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Software, Mutagenesis, Co-Immunoprecipitation Assay, Infection, Knock-Out, Binding Assay, Incubation, Immunoprecipitation

    G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Transfection, Infection, Luciferase, Over Expression, Stable Transfection, Software, Plasmid Preparation

    Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Infection, Luciferase, Software

    G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Transfection, Luciferase, Infection, Software

    G3BP1 targets at upstream of MAVS. a , b Effects of G3BP1 knockdown on the IFN-β promoter and ISRE activation. The control or stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter a or ISRE reporter b (0.1 μg), TK (0.02 μg), and the indicated plasmids (0.1 μg each) for 24 h followed by luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 targets at upstream of MAVS. a , b Effects of G3BP1 knockdown on the IFN-β promoter and ISRE activation. The control or stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter a or ISRE reporter b (0.1 μg), TK (0.02 μg), and the indicated plasmids (0.1 μg each) for 24 h followed by luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Activation Assay, Transfection, Luciferase

    G3BP1 promotes degradation of RNF125 via its auto-ubiquitination. a Interaction between G3BP1 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RNF125 in HEK293T cells. HEK293T cells (5 × 10 7 ) were untreated or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Effects of G3BP1 on the expression of RNF125 or RNF125 (C72/75 A) mutant were evaluated. HEK293T cells were transfected with HA-G3BP1 (0, 0.5, 1.5, and 3 μg) and HA-RNF125 or HA-RNF125 (C72/75 A) plasmids (2 μg) for 24 h. Then the cell lysates were analyzed by immunoblotting with the indicated antibodies. e Effects of SeV infection on the expression of endogenous G3BP1 and RNF125 in HEK293T cells. HEK293T cells were uninfected or infected with SeV for the indicated time. The cell lysates were analyzed by immunoblotting with the indicated antibodies. f Effects of MG132 on G3BP1-mediated destabilization of RNF125. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then the cells were treated with DMSO or MG132 for 6 h before immunoblotting analysis. g Interaction between RNF125 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each). Co-IP and immunoblotting were performed with the indicated antibodies. h Effects of G3BP1 on the interaction between RNF125 and RNF125. The experiments were similarly to those described in g . i Effects of G3BP1 on the ubiquitination of RNF125. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h and then treated with MG132 for 6 h. Co-IP and immunoblotting were performed with the indicated antibodies. j , k Effects of G3BP1 overexpression on RNF125-mediated RIG-I activation were assessed. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE (0.1 μg), HA-RIG-1 (100 ng), Flag-RNF125 (100 ng) or G3BP1 (0, 100, 200, and 400 ng) expression plasmids for 24 h before luciferase assays were performed. The experiment was repeated in triplicates. l Interaction between G3BP1, RIG-I, and RNF125 in HEK293T cells. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. m Interaction between G3BP1, G3BP1 mutants, and RNF125. The experiments were similarly to those described in l . Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV, empty vector, Luc luciferase, αH anti-HA tag, HC heavy chain.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 promotes degradation of RNF125 via its auto-ubiquitination. a Interaction between G3BP1 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RNF125 in HEK293T cells. HEK293T cells (5 × 10 7 ) were untreated or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Effects of G3BP1 on the expression of RNF125 or RNF125 (C72/75 A) mutant were evaluated. HEK293T cells were transfected with HA-G3BP1 (0, 0.5, 1.5, and 3 μg) and HA-RNF125 or HA-RNF125 (C72/75 A) plasmids (2 μg) for 24 h. Then the cell lysates were analyzed by immunoblotting with the indicated antibodies. e Effects of SeV infection on the expression of endogenous G3BP1 and RNF125 in HEK293T cells. HEK293T cells were uninfected or infected with SeV for the indicated time. The cell lysates were analyzed by immunoblotting with the indicated antibodies. f Effects of MG132 on G3BP1-mediated destabilization of RNF125. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then the cells were treated with DMSO or MG132 for 6 h before immunoblotting analysis. g Interaction between RNF125 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each). Co-IP and immunoblotting were performed with the indicated antibodies. h Effects of G3BP1 on the interaction between RNF125 and RNF125. The experiments were similarly to those described in g . i Effects of G3BP1 on the ubiquitination of RNF125. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids for 18 h and then treated with MG132 for 6 h. Co-IP and immunoblotting were performed with the indicated antibodies. j , k Effects of G3BP1 overexpression on RNF125-mediated RIG-I activation were assessed. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE (0.1 μg), HA-RIG-1 (100 ng), Flag-RNF125 (100 ng) or G3BP1 (0, 100, 200, and 400 ng) expression plasmids for 24 h before luciferase assays were performed. The experiment was repeated in triplicates. l Interaction between G3BP1, RIG-I, and RNF125 in HEK293T cells. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. m Interaction between G3BP1, G3BP1 mutants, and RNF125. The experiments were similarly to those described in l . Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV, empty vector, Luc luciferase, αH anti-HA tag, HC heavy chain.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Infection, Expressing, Mutagenesis, Activation Assay, Luciferase, Immunoprecipitation, Plasmid Preparation

    G3BP1 interacts with RIG-I. a G3BP1 interacts with RIG-I but not with MDA5, MAVS, TBK1, or IRF3 in the overexpression system. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg of each) for 24 h. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RIG-I in HEK293T cells. HEK293T cells (5 × 10 7 ) were uninfected or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Domain identification of G3BP1 and RIG-I interaction. HEK293T cells were transfected with the expression plasmids encoding RIG-I and G3BP1 or the corresponding mutants (5 μg each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. e , f Effects of G3BP1 overexpression and its mutants on SeV-triggered IFN-β promoter and ISRE activation. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter (0.1 μg) and the indicated expression plasmids (0.1 μg) for 24 h. Then cells were uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV empty vector, Luc luciferase, αF anti-Flag, αH anti-HA, HC heavy chain, WT wild-type.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 interacts with RIG-I. a G3BP1 interacts with RIG-I but not with MDA5, MAVS, TBK1, or IRF3 in the overexpression system. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg of each) for 24 h. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RIG-I in HEK293T cells. HEK293T cells (5 × 10 7 ) were uninfected or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c , d Domain identification of G3BP1 and RIG-I interaction. HEK293T cells were transfected with the expression plasmids encoding RIG-I and G3BP1 or the corresponding mutants (5 μg each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. e , f Effects of G3BP1 overexpression and its mutants on SeV-triggered IFN-β promoter and ISRE activation. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter (0.1 μg) and the indicated expression plasmids (0.1 μg) for 24 h. Then cells were uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV empty vector, Luc luciferase, αF anti-Flag, αH anti-HA, HC heavy chain, WT wild-type.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Infection, Expressing, Activation Assay, Luciferase, Immunoprecipitation, Plasmid Preparation

    G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Software, Mutagenesis, Co-Immunoprecipitation Assay, Infection, Knock-Out, Binding Assay, Incubation, Immunoprecipitation

    G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 positively regulates SeV-triggered signaling. a – c G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. d – f G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 10 5 ) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. h – j G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Transfection, Infection, Luciferase, Over Expression, Stable Transfection, Software, Plasmid Preparation

    Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: Effects of G3BP1 knockdown on virus-triggered signaling. a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. b – d G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. e – g G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Infection, Luciferase, Software

    G3BP1 positively regulates the cellular antiviral response. a , b G3BP1-overexpressed HEK293T cell lines a or G3BP1-deficient HEK293T cells b were infected with SeV or VSV-GFP (MOI = 0.1) for the indicated time, and then the cell lysates were analyzed by immunoblotting with the antibodies against SeV, GFP, or β-actin. c Effects of G3BP1 on SeV and VSV infection. G3BP1-overexpressed or G3BP1-deficient and control HEK293T cells were infected with SeV for 12 h or with VSV-GFP (MOI = 0.1) for 4 h. The mRNA level of the SeV P and VSV P proteins in cells was determined by qRT-PCR. The experiment was repeated in triplicates. d Effects of G3BP1-overexpressed on VSV titer. G3BP1-overexpressed HEK293T cells were transfected with 1 μg/ml poly (I:C) for 16 h and infected with VSV-GFP (MOI = 0.1) for 18 h. Supernatants were then analyzed for VSV production by standard plaque assays. The experiment was repeated in triplicates. e G3BP1-overexpressed HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 4 h. Images were captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. f Effects of G3BP1-deficient on VSV titer. The experiments were similarly to those described in c . The experiment was repeated in triplicates. g G3BP1-deficient HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 2 h. Images were then captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. qRT-PCR, quantitative real-time polymerase chain reaction. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 positively regulates the cellular antiviral response. a , b G3BP1-overexpressed HEK293T cell lines a or G3BP1-deficient HEK293T cells b were infected with SeV or VSV-GFP (MOI = 0.1) for the indicated time, and then the cell lysates were analyzed by immunoblotting with the antibodies against SeV, GFP, or β-actin. c Effects of G3BP1 on SeV and VSV infection. G3BP1-overexpressed or G3BP1-deficient and control HEK293T cells were infected with SeV for 12 h or with VSV-GFP (MOI = 0.1) for 4 h. The mRNA level of the SeV P and VSV P proteins in cells was determined by qRT-PCR. The experiment was repeated in triplicates. d Effects of G3BP1-overexpressed on VSV titer. G3BP1-overexpressed HEK293T cells were transfected with 1 μg/ml poly (I:C) for 16 h and infected with VSV-GFP (MOI = 0.1) for 18 h. Supernatants were then analyzed for VSV production by standard plaque assays. The experiment was repeated in triplicates. e G3BP1-overexpressed HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 4 h. Images were captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. f Effects of G3BP1-deficient on VSV titer. The experiments were similarly to those described in c . The experiment was repeated in triplicates. g G3BP1-deficient HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 2 h. Images were then captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. qRT-PCR, quantitative real-time polymerase chain reaction. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Infection, Quantitative RT-PCR, Transfection, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling. a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. b – g G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 10 5 ) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i , j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b . Data are mean ± SD of three independent experiments. * P

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Knock-Out, Clone Assay, Generated, CRISPR, Transfection, Luciferase, Infection, Software

    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Journal: Cell Research

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Infection, Real-time Polymerase Chain Reaction