Structured Review

BioLegend inhibitory anti trail antibody
IV-induced macrophage IFN-β upregulates macrophage <t>TRAIL.</t> (A) Murine AM were treated ex vivo with rIFN-β at the given concentrations and TRAIL mRNA expression was quantified and is depicted as fold induction of unstimulated controls. (B) Murine AM were A/PR8-infected (MOI = 0.1) or treated with 180 U/ml rIFN-β ex vivo in presence of a protease inhibitor cocktail to prevent TRAIL shedding and mTRAIL abundance was analysed by FACS after 24 h. Shown are histograms from a representative experiment (top panel) or mean fluorescence intensities (MFI, bottom panel). (C) Murine wt AM were A/PR8 infected at the given MOI and treated with anti-IFN-β Ab, Jak/STAT inhibitor, or DMSO/isotype <t>IgG</t> Ab as control. Murine ifnar −/− AM were A/PR8 infected and left untreated. TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected cells. Bar graphs represent means ± SD of (A) 6; (B) 4 and (C) 5 independent experiments. * p
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1) Product Images from "Macrophage-expressed IFN-? Contributes to Apoptotic Alveolar Epithelial Cell Injury in Severe Influenza Virus Pneumonia"

Article Title: Macrophage-expressed IFN-? Contributes to Apoptotic Alveolar Epithelial Cell Injury in Severe Influenza Virus Pneumonia

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003188

IV-induced macrophage IFN-β upregulates macrophage TRAIL. (A) Murine AM were treated ex vivo with rIFN-β at the given concentrations and TRAIL mRNA expression was quantified and is depicted as fold induction of unstimulated controls. (B) Murine AM were A/PR8-infected (MOI = 0.1) or treated with 180 U/ml rIFN-β ex vivo in presence of a protease inhibitor cocktail to prevent TRAIL shedding and mTRAIL abundance was analysed by FACS after 24 h. Shown are histograms from a representative experiment (top panel) or mean fluorescence intensities (MFI, bottom panel). (C) Murine wt AM were A/PR8 infected at the given MOI and treated with anti-IFN-β Ab, Jak/STAT inhibitor, or DMSO/isotype IgG Ab as control. Murine ifnar −/− AM were A/PR8 infected and left untreated. TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected cells. Bar graphs represent means ± SD of (A) 6; (B) 4 and (C) 5 independent experiments. * p
Figure Legend Snippet: IV-induced macrophage IFN-β upregulates macrophage TRAIL. (A) Murine AM were treated ex vivo with rIFN-β at the given concentrations and TRAIL mRNA expression was quantified and is depicted as fold induction of unstimulated controls. (B) Murine AM were A/PR8-infected (MOI = 0.1) or treated with 180 U/ml rIFN-β ex vivo in presence of a protease inhibitor cocktail to prevent TRAIL shedding and mTRAIL abundance was analysed by FACS after 24 h. Shown are histograms from a representative experiment (top panel) or mean fluorescence intensities (MFI, bottom panel). (C) Murine wt AM were A/PR8 infected at the given MOI and treated with anti-IFN-β Ab, Jak/STAT inhibitor, or DMSO/isotype IgG Ab as control. Murine ifnar −/− AM were A/PR8 infected and left untreated. TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected cells. Bar graphs represent means ± SD of (A) 6; (B) 4 and (C) 5 independent experiments. * p

Techniques Used: Ex Vivo, Expressing, Infection, Protease Inhibitor, FACS, Fluorescence

IV-induced IFN-β is mainly macrophage-derived and induces AEC apoptosis ex vivo and in vivo . (A) C57BL/6 wt mice were infected with 500 pfu A/PR8 and the IFN-α and IFN-β levels (left panel) as well as the amount of infectious virus particles (right panel) were quantified from BALF at indicated time points. (B) AEC apoptosis was quantified in mock or A/PR8 infected mice at d7 pi after intratracheal treatment with IgG isotype Ab, anti IFN-α Ab, anti-IFN-β Ab or both (d5 pi). (C) AEC apoptosis (left panel) and alveolar protein leakage (right panel) were determined at d7 pi after intratracheal treatment with rIFN-β or vehicle (d5 pi). (D) Murine AEC or AM were mock- or A/PR8-infected (live virus or heat-inactivated) ex vivo (MOI = 0.1) and IFN-β release was quantified in supernatants at 24 h pi. (E) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the indicated MOI and IFN-β mRNA expression was quantified at the given times and is depicted as fold induction of mock-infected controls. (F) Uninfected murine AEC were mono- or co-cultured with non-infected AM ex vivo in the presence or absence of IFN-β (180 U/ml) for 24 h, and AEC apoptosis rates were quantified. (G) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the given MOI and TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected controls. (H) Murine AEC or AM were ex vivo infected A/PR8 at the indicated MOI and TRAIL mRNA expression was quantified at the given time and is depicted as fold induction of mock-infected controls. Bar graphs represent means ± SD of (A) 4 animals/group, (B, C) 5 animals/group or of 4 (D, E, G) and 3 (F, H) independent experiments. * p
Figure Legend Snippet: IV-induced IFN-β is mainly macrophage-derived and induces AEC apoptosis ex vivo and in vivo . (A) C57BL/6 wt mice were infected with 500 pfu A/PR8 and the IFN-α and IFN-β levels (left panel) as well as the amount of infectious virus particles (right panel) were quantified from BALF at indicated time points. (B) AEC apoptosis was quantified in mock or A/PR8 infected mice at d7 pi after intratracheal treatment with IgG isotype Ab, anti IFN-α Ab, anti-IFN-β Ab or both (d5 pi). (C) AEC apoptosis (left panel) and alveolar protein leakage (right panel) were determined at d7 pi after intratracheal treatment with rIFN-β or vehicle (d5 pi). (D) Murine AEC or AM were mock- or A/PR8-infected (live virus or heat-inactivated) ex vivo (MOI = 0.1) and IFN-β release was quantified in supernatants at 24 h pi. (E) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the indicated MOI and IFN-β mRNA expression was quantified at the given times and is depicted as fold induction of mock-infected controls. (F) Uninfected murine AEC were mono- or co-cultured with non-infected AM ex vivo in the presence or absence of IFN-β (180 U/ml) for 24 h, and AEC apoptosis rates were quantified. (G) Murine AM were ex vivo infected with live or heat-inactivated A/PR8 at the given MOI and TRAIL mRNA expression was quantified and is depicted as fold induction of mock-infected controls. (H) Murine AEC or AM were ex vivo infected A/PR8 at the indicated MOI and TRAIL mRNA expression was quantified at the given time and is depicted as fold induction of mock-infected controls. Bar graphs represent means ± SD of (A) 4 animals/group, (B, C) 5 animals/group or of 4 (D, E, G) and 3 (F, H) independent experiments. * p

Techniques Used: Derivative Assay, Ex Vivo, In Vivo, Mouse Assay, Infection, Expressing, Cell Culture

2) Product Images from "iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006693

iRhom2 interacts with VISA. (A) Co-immunoprecipitation and immunoblot analysis of HEK293 cells transfected with the indicated plasmids with the indicated antibodies. (B) Detection of the association betweem iRhom2 and VISA by endogenous co-immunoprecipitation and immunoblot analysis with the indicated antibodies in HEK293 cells that were transfected with iRhom2 plasmids and then left un-infected or infected with SeV for the indicated times. (C) Domain mapping of iRhom2-VISA interaction. HEK293 cells were transfected with the indicated truncations before co-immunoprecipitation and immunoblot analysis with the indicated antibodies. The schematic representations of iRhom2 and VISA truncations were shown at the top. (D) Reporter assays for IFNB promoter activity in HEK293 cells that were transfected with iRhom2-shRNA plasmid for 20 h, selected with puromycin (1 μg/ml) and then re-transfected with the IFN-β reporter and indicated plasmids for 24 h. * P
Figure Legend Snippet: iRhom2 interacts with VISA. (A) Co-immunoprecipitation and immunoblot analysis of HEK293 cells transfected with the indicated plasmids with the indicated antibodies. (B) Detection of the association betweem iRhom2 and VISA by endogenous co-immunoprecipitation and immunoblot analysis with the indicated antibodies in HEK293 cells that were transfected with iRhom2 plasmids and then left un-infected or infected with SeV for the indicated times. (C) Domain mapping of iRhom2-VISA interaction. HEK293 cells were transfected with the indicated truncations before co-immunoprecipitation and immunoblot analysis with the indicated antibodies. The schematic representations of iRhom2 and VISA truncations were shown at the top. (D) Reporter assays for IFNB promoter activity in HEK293 cells that were transfected with iRhom2-shRNA plasmid for 20 h, selected with puromycin (1 μg/ml) and then re-transfected with the IFN-β reporter and indicated plasmids for 24 h. * P

Techniques Used: Immunoprecipitation, Transfection, Infection, Activity Assay, shRNA, Plasmid Preparation

iRhom2 suppresses ERAD of VISA. (A) Co-immunoprecipitation and immunoblot analysis using the indicated antibodies with lysates of HEK293T cells transfected with the indicated plasmids. (B) Detection of effects of RNF5, GP78, VCP and HRD1 on the expression of VISA in HEK293T cells transfected with the indicated plasmids by immunoblot analysis with the indicated antibodies. Densitometry quantification was made with ImageJ Software (lower panel) (C) Immunoblot analysis of the indicated proteins in HEK293T cells transfected with the indicated plasmids. (D) IFN-β and ISRE reporter assays with HEK293T cells transfected with the indicated plasmids. (E F) Co-immunoprecipitation and immunoblot analysis using the indicated antibodies with lysates of HEK293T cells transfected with the indicated plasmids.
Figure Legend Snippet: iRhom2 suppresses ERAD of VISA. (A) Co-immunoprecipitation and immunoblot analysis using the indicated antibodies with lysates of HEK293T cells transfected with the indicated plasmids. (B) Detection of effects of RNF5, GP78, VCP and HRD1 on the expression of VISA in HEK293T cells transfected with the indicated plasmids by immunoblot analysis with the indicated antibodies. Densitometry quantification was made with ImageJ Software (lower panel) (C) Immunoblot analysis of the indicated proteins in HEK293T cells transfected with the indicated plasmids. (D) IFN-β and ISRE reporter assays with HEK293T cells transfected with the indicated plasmids. (E F) Co-immunoprecipitation and immunoblot analysis using the indicated antibodies with lysates of HEK293T cells transfected with the indicated plasmids.

Techniques Used: Immunoprecipitation, Transfection, Expressing, Software

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

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

3) Product Images from "Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5"

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

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

4) Product Images from "iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006693

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

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

5) Product Images from "SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007336

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

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

6) Product Images from "ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3"

Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

Journal: Journal of Molecular Cell Biology

doi: 10.1093/jmcb/mjaa004

Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P
Figure Legend Snippet: Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P

Techniques Used: In Vivo, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

7) Product Images from "iRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006693

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

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

8) Product Images from "Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5"

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

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

9) Product Images from "The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0095728

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

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

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

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

10) Product Images from "SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA"

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

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007336

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

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

11) Product Images from "Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5"

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

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

12) Product Images from "Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5"

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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Enzyme-linked Immunosorbent Assay:

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. Enzyme-linked immunosorbent assay The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. RNA quantification Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Article Title: Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus
Article Snippet: .. The homogenates were centrifuged at 5,000 ×g for 5 min at 4°C, and the supernatants were used to measure C3 (ELISA Kit MBS700250, MyBioSource, San Diego, CA, USA), C5 (ELISA Kit MBS704792, MyBioSource), C3a (ELISA Kit MBS70381, MyBioSource), C5a (ELISA Kit MBS700538, MyBioSource), and IFN-β (ELISA Kit 439407, BioLegend). .. TLR-4 was measured by flow cytometry in cells obtained from fresh spleens, which were disaggregated and passed through a 70 μ m nylon mesh.

Article Title: The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages
Article Snippet: .. IFN-β detection in cell culture medium Murine IFN-β was analyzed using the Legend Max Mouse IFN-β ELISA kit with pre-coated plates (BioLegend, San Diego, California, USA); human IFN-β was analyzed using the VeriKine human IFN-β ELISA kit (PBL Interferon Source, Piscataway, New Jersey, USA). .. Detection was performed by light absorbance measurement at a wavelength of 450 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, Vermont, USA).

Article Title: Deficiency of the AIM2–ASC Signal Uncovers the STING-Driven Overreactive Response of Type I IFN and Reciprocal Depression of Protective IFN-γ Immunity in Mycobacterial Infection
Article Snippet: .. Murine IFN-β (439408; BioLegend) and mouse IFN-r (P105796; R & D Systems) ELISA kits were used. .. All ASC truncations were generated by PCR and subcloned into a pcDNA3 vector.

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Concentration Assay:

Article Title: POL7085 or anti‐CCL28 treatment inhibits development of post‐paramyxoviral airway disease
Article Snippet: .. Using commercial ELISAs the concentration of IL‐9 (eBioscience, 88–8092, sensitivity 32 pg/mL), IL‐10 (BioLegend, 431418, sensitivity 2.7 pg/mL), IL‐13 (eBioscience, BMS6015, sensitivity 2.8 pg/mL), IL‐33 (eBioscience, 88–7333, sensitivity 25 pg/mL), IFNα (eBioscience, BMS6027, sensitivity 7.48 pg/mL), IFNβ (BioLegend, 439407, sensitivity 1.9 pg/mL), and IFN‐γ (eBioscience, BMS606, sensitivity 5.3 pg/mL) in the BAL was determined according to manufacturer's instructions. ..

Cell Culture:

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. Enzyme-linked immunosorbent assay The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. RNA quantification Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Article Title: The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages
Article Snippet: .. IFN-β detection in cell culture medium Murine IFN-β was analyzed using the Legend Max Mouse IFN-β ELISA kit with pre-coated plates (BioLegend, San Diego, California, USA); human IFN-β was analyzed using the VeriKine human IFN-β ELISA kit (PBL Interferon Source, Piscataway, New Jersey, USA). .. Detection was performed by light absorbance measurement at a wavelength of 450 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski, Vermont, USA).

Article Title: The cGas–Sting Signaling Pathway Is Required for the Innate Immune Response Against Ectromelia Virus
Article Snippet: .. Cell culture supernatants and mouse serum were collected, and levels of mouse IFN-β (439407, BioLegend) were measured according to manufacturer’s instructions. .. For the preparation of soluble cell extracts, harvested cells were washed two times with cold phosphate-buffered saline (PBS) and then were lysed in RIPA buffer containing protease and phosphatase inhibitors (P0013, Beyotime Biotechnology, China).

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Infection:

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. Enzyme-linked immunosorbent assay The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. RNA quantification Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Mouse Assay:

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. Enzyme-linked immunosorbent assay The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. RNA quantification Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

Article Title: MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation
Article Snippet: .. The concentrations of cytokines in cell culture supernatants and sera from VSV infected mice were measured using mouse IFN-β (439407, BioLegend) and IL-6 (431301, BioLegend), mouse TNF-α (88-7324-88, eBioscience) enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacturer’s instructions. .. Total RNA was extracted with TRIzol reagent following the manufacturer’s instructions (Invitrogen).

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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: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend murine ifn β pbl
    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 Pbl, supplied by BioLegend, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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