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Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the <t>IFN-β</t> promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
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Article Title: Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161015

Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
Figure Legend Snippet: Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

Techniques Used: Activation Assay, Transfection, Luciferase, Mutagenesis, De-Phosphorylation Assay, Infection, Immunoprecipitation

Desumoylation of RIG-I and MDA5 by SENP2 at the late phase of viral infection. (A) Effects of SENPs on sumoylation of RIG-I and MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h before Ni 2+ pull-down assays and immunoblotting analysis. (B) Effects of knockdown of SENP1 and SENP2 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 36 h, and then infected with SeV for 10 h or transfected with poly(I:C) for 18 h before luciferase assays. The knockdown efficiencies of SENP1 and SENP2 shRNAs are shown at the right panels. HEK293T cells were transfected with the indicated plasmids for 24 h followed by immunoblotting analysis. (C) Effects of SENP2 deficiency on RIG-I and MDA5-mediated activation of TBK1. The indicated proteins were transduced into SENP2- and vector-reconstituted SENP2 −/− MEFs via retroviral approach, and then cells were harvested, followed by immunoblotting analysis with the indicated antibodies. (D and E) Effects of SENP2 deficiency on sumoylation and K48-linked polyubiquitination of RIG-I and MDA5. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV (D) or EMCV (E) for the indicated times, followed by immunoprecipitation and immunoblotting analysis. (F) Effects of SENP2 deficiency on SeV- and EMCV-induced transcription of downstream antiviral genes. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times before qPCR analysis. (G) Effects of viral infection and poly(I:C)-transfected on expression of SENP2. Cells were infected with SeV (left) or transfected with poly(I:C) (right) for the indicated times before lysed for immunoblotting analysis with the indicated antibodies. Data in B and F are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
Figure Legend Snippet: Desumoylation of RIG-I and MDA5 by SENP2 at the late phase of viral infection. (A) Effects of SENPs on sumoylation of RIG-I and MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h before Ni 2+ pull-down assays and immunoblotting analysis. (B) Effects of knockdown of SENP1 and SENP2 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 36 h, and then infected with SeV for 10 h or transfected with poly(I:C) for 18 h before luciferase assays. The knockdown efficiencies of SENP1 and SENP2 shRNAs are shown at the right panels. HEK293T cells were transfected with the indicated plasmids for 24 h followed by immunoblotting analysis. (C) Effects of SENP2 deficiency on RIG-I and MDA5-mediated activation of TBK1. The indicated proteins were transduced into SENP2- and vector-reconstituted SENP2 −/− MEFs via retroviral approach, and then cells were harvested, followed by immunoblotting analysis with the indicated antibodies. (D and E) Effects of SENP2 deficiency on sumoylation and K48-linked polyubiquitination of RIG-I and MDA5. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV (D) or EMCV (E) for the indicated times, followed by immunoprecipitation and immunoblotting analysis. (F) Effects of SENP2 deficiency on SeV- and EMCV-induced transcription of downstream antiviral genes. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times before qPCR analysis. (G) Effects of viral infection and poly(I:C)-transfected on expression of SENP2. Cells were infected with SeV (left) or transfected with poly(I:C) (right) for the indicated times before lysed for immunoblotting analysis with the indicated antibodies. Data in B and F are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

Techniques Used: Infection, Transfection, Activation Assay, Luciferase, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

TRIM38 is required for RLR-mediated innate immune response. (A and B) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDMs. The indicated cells were left uninfected or infected with the indicated viruses for 6 h (A) or infected with SeV or EMCV for the indicated times (B) before qPCR analysis. (C) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDCs or MLFs. Trim38 +/+ and Trim38 −/− BMDCs (A) or MLFs (B) were left uninfected or infected with EMCV, VSV, or SeV for 6 h before qPCR analysis. (D) Effects of Trim38 deficiency on EMCV- and SeV-induced secretion of Ifn-β and Tnfα cytokines in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for 18 h before ELISA with the culture medium. (E) Effects of Trim38 deficiency on EMCV- or SeV-induced phosphorylation of Tbk1, Irf3, and IκBα in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for the indicated times, followed by immunoblotting analysis. (F) Effects of Trim38 deficiency on transcription of antiviral genes induced by transfected nucleic acids in MLFs. The indicated cells were transfected with the indicated nucleic acids for 6 h before qPCR analysis. (G) Effects of Trim38 deficiency on IFN-α4- or IFN-β–induced transcription of Cxcl10 in BMDMs. The indicated cells were left untreated or treated with IFN-α4 or IFN-β for the indicated times for the indicated times before qPCR analysis. (H) Effects of Trim38 deficiency on VSV- or EMCV-induced death of mice. Trim38 +/+ and Trim38 −/− mice ( n = 16) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse, and the survival rates of mice were observed and recorded for two weeks. (I) Measurement of viral titers in the brain of infected mice. Trim38 +/+ and Trim38 −/− mice ( n = 3) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse. 2 d later, the brains of the infected mice were extracted for measurement of viral titers. The P-values were calculated using the Student’s t test. Data in A, B, and C are from four biological replicates. Data in F and G are from one representative experiment with three technical replicates. The error bars are mean ± SD in A–D, F, G, and I. Experiments were repeated twice (E–I) or three times (D).
Figure Legend Snippet: TRIM38 is required for RLR-mediated innate immune response. (A and B) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDMs. The indicated cells were left uninfected or infected with the indicated viruses for 6 h (A) or infected with SeV or EMCV for the indicated times (B) before qPCR analysis. (C) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDCs or MLFs. Trim38 +/+ and Trim38 −/− BMDCs (A) or MLFs (B) were left uninfected or infected with EMCV, VSV, or SeV for 6 h before qPCR analysis. (D) Effects of Trim38 deficiency on EMCV- and SeV-induced secretion of Ifn-β and Tnfα cytokines in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for 18 h before ELISA with the culture medium. (E) Effects of Trim38 deficiency on EMCV- or SeV-induced phosphorylation of Tbk1, Irf3, and IκBα in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for the indicated times, followed by immunoblotting analysis. (F) Effects of Trim38 deficiency on transcription of antiviral genes induced by transfected nucleic acids in MLFs. The indicated cells were transfected with the indicated nucleic acids for 6 h before qPCR analysis. (G) Effects of Trim38 deficiency on IFN-α4- or IFN-β–induced transcription of Cxcl10 in BMDMs. The indicated cells were left untreated or treated with IFN-α4 or IFN-β for the indicated times for the indicated times before qPCR analysis. (H) Effects of Trim38 deficiency on VSV- or EMCV-induced death of mice. Trim38 +/+ and Trim38 −/− mice ( n = 16) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse, and the survival rates of mice were observed and recorded for two weeks. (I) Measurement of viral titers in the brain of infected mice. Trim38 +/+ and Trim38 −/− mice ( n = 3) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse. 2 d later, the brains of the infected mice were extracted for measurement of viral titers. The P-values were calculated using the Student’s t test. Data in A, B, and C are from four biological replicates. Data in F and G are from one representative experiment with three technical replicates. The error bars are mean ± SD in A–D, F, G, and I. Experiments were repeated twice (E–I) or three times (D).

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

TRIM38 positively regulates RIG-I– and MDA5-mediated signaling. (A) Coimmunoprecipitation of TRIM38 with MDA5 and RIG-I in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation experiments and immunoblotting analysis. (B) Endogenous association of TRIM38 with RIG-I and MDA5. THP-1 cells were left uninfected or infected with SeV (top) or EMCV (bottom) for the indicated times followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (C and D) Domain mapping of TRIM38 with RIG-I and MDA5. Experiments were performed as in B, except for the different transfected plasmids. (E) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (F) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated cellular antiviral response. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by VSV (MOI = 0.1) infection for 24 h, and then the supernatants were collected for plaque assays to determine the viral titers. (G) Effects of knockdown of TRIM38 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. (left) HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. (right) HEK293 cells were transfected with the indicated plasmids for 36 h, and then transfected with infected with SeV or poly(I:C) for 18 h for 10 h before luciferase assays. (H) Effects of TRIM38 knockdown on SeV- or poly(I:C)-induced transcription of downstream antiviral genes. HEK293 cells were transfected and treated as in (I) before qPCR analysis. Data are from one representative experiment with four (E and G) or three (H) technical replicates. The error bars are mean ± SD in E, G, and H. All experiments were repeated twice.
Figure Legend Snippet: TRIM38 positively regulates RIG-I– and MDA5-mediated signaling. (A) Coimmunoprecipitation of TRIM38 with MDA5 and RIG-I in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation experiments and immunoblotting analysis. (B) Endogenous association of TRIM38 with RIG-I and MDA5. THP-1 cells were left uninfected or infected with SeV (top) or EMCV (bottom) for the indicated times followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (C and D) Domain mapping of TRIM38 with RIG-I and MDA5. Experiments were performed as in B, except for the different transfected plasmids. (E) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (F) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated cellular antiviral response. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by VSV (MOI = 0.1) infection for 24 h, and then the supernatants were collected for plaque assays to determine the viral titers. (G) Effects of knockdown of TRIM38 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. (left) HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. (right) HEK293 cells were transfected with the indicated plasmids for 36 h, and then transfected with infected with SeV or poly(I:C) for 18 h for 10 h before luciferase assays. (H) Effects of TRIM38 knockdown on SeV- or poly(I:C)-induced transcription of downstream antiviral genes. HEK293 cells were transfected and treated as in (I) before qPCR analysis. Data are from one representative experiment with four (E and G) or three (H) technical replicates. The error bars are mean ± SD in E, G, and H. All experiments were repeated twice.

Techniques Used: Over Expression, Transfection, Infection, Activation Assay, Luciferase, Real-time Polymerase Chain Reaction

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

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    BioLegend murine ifn β
    Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the <t>IFN-β</t> promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.
    Murine Ifn β, supplied by BioLegend, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    murine ifn β - by Bioz Stars, 2020-01
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    Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20161015

    Figure Lengend Snippet: Sumoylation of the CARDs of MDA5 and RIG-I is critical for their recruitment of PP1α. (A) Effects of MDA5, RIG-I, and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (B) Effects of sumoylation-defective mutation of the CARDs of RIG-I and MDA5 on their dephosphorylation, K63-linked polyubiquitination and recruitment of PP1α after viral infection. The reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times, followed by immunoprecipitation. The immunoprecipitates were divided into two equal portions, and one was used for immunoblot analysis with the PP1α antibody and the other was lysed in denaturing conditions and reimmunoprecipitated for endogenous ubiquitination detection. (C) Effects of sumoylation of RIG-I-CARD or MDA5-CARD on their interactions with PP1α. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation and immunoblotting analysis. (D) Effects of RIG-I-, MDA5- and their mutants on activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h followed by luciferase assays. Data in A and d are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

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

    Techniques: Activation Assay, Transfection, Luciferase, Mutagenesis, De-Phosphorylation Assay, Infection, Immunoprecipitation

    Desumoylation of RIG-I and MDA5 by SENP2 at the late phase of viral infection. (A) Effects of SENPs on sumoylation of RIG-I and MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h before Ni 2+ pull-down assays and immunoblotting analysis. (B) Effects of knockdown of SENP1 and SENP2 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 36 h, and then infected with SeV for 10 h or transfected with poly(I:C) for 18 h before luciferase assays. The knockdown efficiencies of SENP1 and SENP2 shRNAs are shown at the right panels. HEK293T cells were transfected with the indicated plasmids for 24 h followed by immunoblotting analysis. (C) Effects of SENP2 deficiency on RIG-I and MDA5-mediated activation of TBK1. The indicated proteins were transduced into SENP2- and vector-reconstituted SENP2 −/− MEFs via retroviral approach, and then cells were harvested, followed by immunoblotting analysis with the indicated antibodies. (D and E) Effects of SENP2 deficiency on sumoylation and K48-linked polyubiquitination of RIG-I and MDA5. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV (D) or EMCV (E) for the indicated times, followed by immunoprecipitation and immunoblotting analysis. (F) Effects of SENP2 deficiency on SeV- and EMCV-induced transcription of downstream antiviral genes. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times before qPCR analysis. (G) Effects of viral infection and poly(I:C)-transfected on expression of SENP2. Cells were infected with SeV (left) or transfected with poly(I:C) (right) for the indicated times before lysed for immunoblotting analysis with the indicated antibodies. Data in B and F are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20161015

    Figure Lengend Snippet: Desumoylation of RIG-I and MDA5 by SENP2 at the late phase of viral infection. (A) Effects of SENPs on sumoylation of RIG-I and MDA5. HEK293 cells were transfected with the indicated plasmids for 24 h before Ni 2+ pull-down assays and immunoblotting analysis. (B) Effects of knockdown of SENP1 and SENP2 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 36 h, and then infected with SeV for 10 h or transfected with poly(I:C) for 18 h before luciferase assays. The knockdown efficiencies of SENP1 and SENP2 shRNAs are shown at the right panels. HEK293T cells were transfected with the indicated plasmids for 24 h followed by immunoblotting analysis. (C) Effects of SENP2 deficiency on RIG-I and MDA5-mediated activation of TBK1. The indicated proteins were transduced into SENP2- and vector-reconstituted SENP2 −/− MEFs via retroviral approach, and then cells were harvested, followed by immunoblotting analysis with the indicated antibodies. (D and E) Effects of SENP2 deficiency on sumoylation and K48-linked polyubiquitination of RIG-I and MDA5. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV (D) or EMCV (E) for the indicated times, followed by immunoprecipitation and immunoblotting analysis. (F) Effects of SENP2 deficiency on SeV- and EMCV-induced transcription of downstream antiviral genes. Senp2 −/− or SENP2-reconstituted MEFs were left uninfected or infected with SeV or EMCV for the indicated times before qPCR analysis. (G) Effects of viral infection and poly(I:C)-transfected on expression of SENP2. Cells were infected with SeV (left) or transfected with poly(I:C) (right) for the indicated times before lysed for immunoblotting analysis with the indicated antibodies. Data in B and F are from one representative experiment with three technical replicates (mean ± SD). All the experiments were repeated three times.

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

    Techniques: Infection, Transfection, Activation Assay, Luciferase, Plasmid Preparation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

    TRIM38 is required for RLR-mediated innate immune response. (A and B) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDMs. The indicated cells were left uninfected or infected with the indicated viruses for 6 h (A) or infected with SeV or EMCV for the indicated times (B) before qPCR analysis. (C) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDCs or MLFs. Trim38 +/+ and Trim38 −/− BMDCs (A) or MLFs (B) were left uninfected or infected with EMCV, VSV, or SeV for 6 h before qPCR analysis. (D) Effects of Trim38 deficiency on EMCV- and SeV-induced secretion of Ifn-β and Tnfα cytokines in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for 18 h before ELISA with the culture medium. (E) Effects of Trim38 deficiency on EMCV- or SeV-induced phosphorylation of Tbk1, Irf3, and IκBα in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for the indicated times, followed by immunoblotting analysis. (F) Effects of Trim38 deficiency on transcription of antiviral genes induced by transfected nucleic acids in MLFs. The indicated cells were transfected with the indicated nucleic acids for 6 h before qPCR analysis. (G) Effects of Trim38 deficiency on IFN-α4- or IFN-β–induced transcription of Cxcl10 in BMDMs. The indicated cells were left untreated or treated with IFN-α4 or IFN-β for the indicated times for the indicated times before qPCR analysis. (H) Effects of Trim38 deficiency on VSV- or EMCV-induced death of mice. Trim38 +/+ and Trim38 −/− mice ( n = 16) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse, and the survival rates of mice were observed and recorded for two weeks. (I) Measurement of viral titers in the brain of infected mice. Trim38 +/+ and Trim38 −/− mice ( n = 3) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse. 2 d later, the brains of the infected mice were extracted for measurement of viral titers. The P-values were calculated using the Student’s t test. Data in A, B, and C are from four biological replicates. Data in F and G are from one representative experiment with three technical replicates. The error bars are mean ± SD in A–D, F, G, and I. Experiments were repeated twice (E–I) or three times (D).

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20161015

    Figure Lengend Snippet: TRIM38 is required for RLR-mediated innate immune response. (A and B) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDMs. The indicated cells were left uninfected or infected with the indicated viruses for 6 h (A) or infected with SeV or EMCV for the indicated times (B) before qPCR analysis. (C) Effects of Trim38 deficiency on RNA virus–induced transcription of downstream antiviral genes in BMDCs or MLFs. Trim38 +/+ and Trim38 −/− BMDCs (A) or MLFs (B) were left uninfected or infected with EMCV, VSV, or SeV for 6 h before qPCR analysis. (D) Effects of Trim38 deficiency on EMCV- and SeV-induced secretion of Ifn-β and Tnfα cytokines in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for 18 h before ELISA with the culture medium. (E) Effects of Trim38 deficiency on EMCV- or SeV-induced phosphorylation of Tbk1, Irf3, and IκBα in BMDMs. The indicated cells were left uninfected or infected with EMCV or SeV for the indicated times, followed by immunoblotting analysis. (F) Effects of Trim38 deficiency on transcription of antiviral genes induced by transfected nucleic acids in MLFs. The indicated cells were transfected with the indicated nucleic acids for 6 h before qPCR analysis. (G) Effects of Trim38 deficiency on IFN-α4- or IFN-β–induced transcription of Cxcl10 in BMDMs. The indicated cells were left untreated or treated with IFN-α4 or IFN-β for the indicated times for the indicated times before qPCR analysis. (H) Effects of Trim38 deficiency on VSV- or EMCV-induced death of mice. Trim38 +/+ and Trim38 −/− mice ( n = 16) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse, and the survival rates of mice were observed and recorded for two weeks. (I) Measurement of viral titers in the brain of infected mice. Trim38 +/+ and Trim38 −/− mice ( n = 3) were intranasally infected with VSV at 10 8 PFU per mouse or EMCV at 10 5 PFU per mouse. 2 d later, the brains of the infected mice were extracted for measurement of viral titers. The P-values were calculated using the Student’s t test. Data in A, B, and C are from four biological replicates. Data in F and G are from one representative experiment with three technical replicates. The error bars are mean ± SD in A–D, F, G, and I. Experiments were repeated twice (E–I) or three times (D).

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

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

    TRIM38 positively regulates RIG-I– and MDA5-mediated signaling. (A) Coimmunoprecipitation of TRIM38 with MDA5 and RIG-I in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation experiments and immunoblotting analysis. (B) Endogenous association of TRIM38 with RIG-I and MDA5. THP-1 cells were left uninfected or infected with SeV (top) or EMCV (bottom) for the indicated times followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (C and D) Domain mapping of TRIM38 with RIG-I and MDA5. Experiments were performed as in B, except for the different transfected plasmids. (E) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (F) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated cellular antiviral response. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by VSV (MOI = 0.1) infection for 24 h, and then the supernatants were collected for plaque assays to determine the viral titers. (G) Effects of knockdown of TRIM38 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. (left) HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. (right) HEK293 cells were transfected with the indicated plasmids for 36 h, and then transfected with infected with SeV or poly(I:C) for 18 h for 10 h before luciferase assays. (H) Effects of TRIM38 knockdown on SeV- or poly(I:C)-induced transcription of downstream antiviral genes. HEK293 cells were transfected and treated as in (I) before qPCR analysis. Data are from one representative experiment with four (E and G) or three (H) technical replicates. The error bars are mean ± SD in E, G, and H. All experiments were repeated twice.

    Journal: The Journal of Experimental Medicine

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

    doi: 10.1084/jem.20161015

    Figure Lengend Snippet: TRIM38 positively regulates RIG-I– and MDA5-mediated signaling. (A) Coimmunoprecipitation of TRIM38 with MDA5 and RIG-I in mammalian overexpression system. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by coimmunoprecipitation experiments and immunoblotting analysis. (B) Endogenous association of TRIM38 with RIG-I and MDA5. THP-1 cells were left uninfected or infected with SeV (top) or EMCV (bottom) for the indicated times followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (C and D) Domain mapping of TRIM38 with RIG-I and MDA5. Experiments were performed as in B, except for the different transfected plasmids. (E) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated activation of the IFN-β promoter. HEK293 cells were transfected with the indicated plasmids for 24 h before luciferase assays. (F) Effects of TRIM38 or TRIM38(C31S) on RIG-I– and MDA5-mediated cellular antiviral response. HEK293 cells were transfected with the indicated plasmids for 24 h, followed by VSV (MOI = 0.1) infection for 24 h, and then the supernatants were collected for plaque assays to determine the viral titers. (G) Effects of knockdown of TRIM38 on SeV- or poly(I:C)-induced activation of the IFN-β promoter. (left) HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis. (right) HEK293 cells were transfected with the indicated plasmids for 36 h, and then transfected with infected with SeV or poly(I:C) for 18 h for 10 h before luciferase assays. (H) Effects of TRIM38 knockdown on SeV- or poly(I:C)-induced transcription of downstream antiviral genes. HEK293 cells were transfected and treated as in (I) before qPCR analysis. Data are from one representative experiment with four (E and G) or three (H) technical replicates. The error bars are mean ± SD in E, G, and H. All experiments were repeated twice.

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

    Techniques: Over Expression, Transfection, Infection, Activation Assay, Luciferase, Real-time Polymerase Chain Reaction