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rabbit anti-neurl3  (Millipore)

 
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    Structured Review

    Millipore rabbit anti-neurl3
    <t>NEURL3</t> is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Rabbit Anti Neurl3, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein"

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01123-18

    NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Infection, Microarray, Quantitative RT-PCR, Knock-Out

    NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.
    Figure Legend Snippet: NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.

    Techniques Used: Quantitative RT-PCR

    NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Luciferase, Transfection

    NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).
    Figure Legend Snippet: NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).

    Techniques Used: Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.
    Figure Legend Snippet: Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot

    NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.
    Figure Legend Snippet: NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Immunofluorescence, Staining

    NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.
    Figure Legend Snippet: NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.

    Techniques Used: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Immunoprecipitation

    NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.
    Figure Legend Snippet: NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Techniques Used: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, Immunofluorescence, Staining

    NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.
    Figure Legend Snippet: NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.

    Techniques Used: Infection, Plasmid Preparation, Quantitative RT-PCR, Titration, Expressing, Immunoprecipitation, Western Blot

    The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Activity Assay, Functional Assay, Expressing, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Knock-Out, Infection, Amplification, Western Blot, Expressing, Transduction, Quantitative RT-PCR, Titration

    Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).
    Figure Legend Snippet: Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).

    Techniques Used: Infection, Expressing, Microarray, RNA Sequencing Assay



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    90
    Millipore rabbit anti-neurl3
    <t>NEURL3</t> is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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    NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 is induced by HCV infection. (A) NEURL3 was upregulated in the mRNA microarray analysis of Huh7-MAVSR cells infected with HCVcc (JFH1 strain) at an MOI of 5 at 24, 48, and 72 h postinfection. (B and C) RT-qPCR analysis of NEURL3, IFN-β, and HCV RNA levels in Huh7-MAVSR cells infected with HCVcc at an MOI of 5 at 24, 48, and 72 h postinfection (B) and in primary human hepatocytes infected with HCVcc at an MOI of 2 at 48 h postinfection (C). Values represent means ± standard deviations (SD) from 2 independent experiments. (D to F) RT-qPCR analysis of NEURL3 (D), IFN-β (E), and HCV (F) RNA levels in the MDA5 knockout Huh7-MAVSR cells as well as control cells that were infected with HCVcc at an MOI of 5. (G to I) The LGP2 knockout Huh7-MAVSR cells as well as control cells were infected with HCVcc at an MOI of 5. The cells were collected at the indicated times postinfection and analyzed by RT-qPCR for the intracellular NEURL3 (G), IFN-β (H), and HCV (I) RNA levels. Values are presented as mRNA fold induction relative to the noninfection control and represent means ± SD from 3 independent experiments. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Microarray, Quantitative RT-PCR, Knock-Out

    NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 is not an IFN-stimulated gene. (A to D) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7-MAVSR cells treated with 100 IU/ml of IFN-α (A), 100 IU/ml of IFN-β (B), 100 ng/ml of IFN-γ (C), or 100 ng/ml of IFN-λ3 (IL-28B) (D). (E to H) RT-qPCR analysis of NEURL3 and ISG (MXA or IP-10) mRNA levels in Huh7 cells treated with 100 IU/ml of IFN-α (E), 100 IU/ml of IFN-β (F), 100 ng/ml of IFN-γ (G), or 100 ng/ml of IFN-λ3 (IL-28B) (H). Values are presented as fold induction relative to the mock control and represent means ± SD from 2 independent experiments.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Quantitative RT-PCR

    NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 inhibits HCV infection. (A) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3 or empty vector control. (B and C) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels were determined by RT-qPCR (B), and the infectivity titers in the culture supernatants were determined by titration assay (C) at 24, 48, 72, and 96 h postinfection. Assays were performed in duplicates, and error bars indicate the means ± SD. (D) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVpp (strain H77). The infection was quantified by luciferase assay. Assays were performed in triplicates, and error bars indicate the means ± SD. (E) JFH-1 subgenomic replicon cells were transfected with NEURL3-expressing plasmid, and intracellular HCV NS3 and NEURL3 protein levels at 24, 48, and 72 h posttransfection were analyzed by Western blotting. (F) RT-qPCR analysis of Huh7-Vector or Huh7-NEURL3 cells that were infected with HCVΔE1 virus (JFH1 with the E1 deletion) (36), and intracellular HCV RNA levels at day 3 postinfection were analyzed by RT-qPCR and normalized to the cellular actin mRNA levels. Assays were performed in duplicates, and error bars indicate the means ± SD. (G to I) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc at an MOI of 2, and intracellular HCV RNA (G), extracellular infectivity titers (H), and intracellular infectivity titers (I) at 24 h postinfection were determined by RT-qPCR or titration assay, respectively. Assays were performed in triplicates, and error bars indicate the means ± SD. ns, non significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Luciferase, Transfection

    NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 has no antiviral effect against ZIKV, DENV, and VSV. Huh7-Vector or Huh7-NEURL3 cells were infected with ZIKV (A to C) or DENV (D to F) at an MOI of 0.1 or with VSV (G to I) at an MOI of 1. The intracellular (A, D, G) and extracellular (B, E, H) RNA levels as well as extracellular virus titers (C, F, I) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and plaque/titration assay, respectively. Assays were performed in duplicate or triplicate, and error bars indicate the means ± SD. ns, not significant (P > 0.05).

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Interaction between NEURL3 and HCV structural and nonstructural proteins. HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 (A), core (B), E2 (C), NS2 (D), NS3 (E), NS4B (F), NS5A (G), or NS5B (H), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. (I) HEK293T cells were cotransfected with plasmids expressing E1-A4 and NEURL3-Flag, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-A4 and anti-Flag antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunoprecipitation, Western Blot

    NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 specifically colocalizes with HCV E1 protein. (A) Huh7-Vector or Huh7-NEURL3-Flag cells were transfected with plasmids expressing E1, E2, or NS2 for 48 h and subjected to immunofluorescence staining of NEURL3 (green) and E1, E2, or NS2 (red). The colocalization of NEURL3 and HCV protein was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E1, E2, or NS2) and the y axis representing green signal (NEURL3). (B) Pearson's coefficient, the indicator of colocalization between NEURL3 and HCV protein (E1, E2, and NS2), was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Plasmid Preparation, Transfection, Expressing, Immunofluorescence, Staining

    NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 interacts with HCV envelope glycoprotein E1 in the context of HCV infection. (A) Western blot of Huh7 cells that were transduced with lentivirus expressing Flag-tagged NEURL3 or empty vector control. The anti-Flag antibody was used in the blot. (B, C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 0.01. The intracellular HCV RNA levels (B) and extracellular infectivity titers (C) at day 6 postinfection were determined by RT-qPCR and titration assay. Assays were performed in duplicates, and error bars indicate the mean ± SD. **, P < 0.01; ***, P < 0.001. (D) Huh7 cells expressing NEURL3-Flag or empty vector control were infected with JFH1 virus containing an A4 epitope in E1 at an MOI of 1. Cell lysates collected at day 7 postinfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting using anti-A4, anti-Flag, anti-core, and anti-E2 antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Western Blot, Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Titration, Immunoprecipitation

    NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 blocks the E1 and E2 interaction. (A) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1, E2, and NEURL3, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-E2 and anti-Flag antibodies. (B) Quantification of coimmunoprecipitated E2 relative to input E2 by Image J. The values were normalized to coimmunoprecipitated E1 and expressed as a relative value to the control. Error bars indicate the means ± SD from 3 individual experiments. ***, P < 0.001. (C) Huh7-Vector or Huh7-NEURL3-Flag cells were infected with HCVcc (strain JFH1-E1A4) at an MOI of 1 for 48 h and subjected to immunofluorescence staining of NEURL3 (blue), E1 (green), and E2 (red). The colocalization of E1 and E2 was analyzed in pixel dot plots on the right, with the x axis representing red signal intensity (E2) and the y axis representing green signal (E1). (D) Pearson's coefficient, the indicator of colocalization between E1 and E2, was determined by regression of the pixel dot plots. Ten individual images were analyzed for each group. Error bars indicate the means ± SD. ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Infection, Immunofluorescence, Staining

    NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: NEURL3 inhibits HCV infection of different genotypes. (A to F) Huh7-Vector or Huh7-NEURL3 cells were infected with HCVcc of strain Con1 (genotype 1b), strain S52 (genotype 3a), and strain HK6a (genotype 6a) at an MOI of 0.01. The extracellular infectivity titers (A to C) and intracellular HCV RNA levels (D to F) at different time points were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G to I) HEK293T cells were cotransfected with plasmids expressing NEURL3 together with Flag-tagged E1 of Con1 (G), S52 (H), or HK6a (I), and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Plasmid Preparation, Quantitative RT-PCR, Titration, Expressing, Immunoprecipitation, Western Blot

    The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: The NHR domain but not the RING domain of NEURL3 is required for its anti-HCV activity. (A) Schematic presentation of functional domains of NEURL3 and the mutants used in the study. (B) HEK293T cells were cotransfected with plasmids expressing Flag-tagged E1 and NEURL3, NEURL3-ΔRING, or NEURL3-ΔNHR, and cell lysates collected at day 2 posttransfection were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates and inputs were analyzed by Western blotting with anti-NEURL3 and anti-Flag antibodies. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (C) Western blotting of Huh7 cells that were transduced with lentivirus expressing NEURL3, NEURL3-ΔRING, NEURL3-ΔNHR, or empty vector control. The asterisk indicates a smaller protein associated with NEURL3-ΔRING. (D, E) Huh7-Vector, Huh7-NEURL3, Hnu7-NEURL3-ΔRING, or Huh7-NEURL3-ΔNHR cells were infected with HCVcc (strain JFH1) at an MOI of 0.01. The intracellular HCV RNA levels (D) and supernatant infectivity titers (E) at 24, 48, 72, and 96 h postinfection were determined by RT-qPCR and titration assay, respectively. Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Activity Assay, Functional Assay, Expressing, Immunoprecipitation, Western Blot, Transduction, Plasmid Preparation, Infection, Quantitative RT-PCR, Titration

    Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Knockout of NEURL3 promotes HCV infection. (A) Schematic illustration of the NEURL3-sgRNA targeting sites on the genome. (B) Alignment of the subcloned NEURL3 mutations in NEURL3 knockout Huh7-MAVSR cells. The sequences spanning the NEURL3-sgRNA targeting sites in the knockout cells were amplified by PCR and subcloned. The sequences of 8 subclones are presented. The designed sgRNA targeting sequences are marked with a box. (C to E) NEURL3 knockout or control Huh7-MAVSR cells were infected with HCVcc (strain JFH1) at an MOI of 1, and the intracellular HCV RNA levels (C), infectivity titers (D), and NEURL3 mRNA levels (E) at 24, 48, 72, and 96 h postinfection were determined. Assays were performed in duplicates, and error bars indicate the means ± SD. (F) Sequences of the sgRNA-resistant NEURL3. The introduced mutations change only nucleotide sequences but not amino acid sequences. (G) Western blot analysis of Huh7-MAVSR cells in which NEURL3 knockout was rescued by exogenous expression of sgRNA-resistant NEURL3 using an anti-NEURL3 antibody. (H, I) The NEURL3 knockout Huh7-MAVSR cells were transduced with lentivirus expressing sgRNA-resistant NEURL3 to rescue the NEURL3 expression. These cells were infected with HCVcc (strain JFH1) at an MOI of 1 and then collected at 24, 48, 72, and 96 h postinfection to determine intracellular HCV RNA by RT-qPCR (H) or supernatant infectivity titers by titration assay (I). Assays were performed in duplicates, and error bars indicate the means ± SD. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Knock-Out, Infection, Amplification, Western Blot, Expressing, Transduction, Quantitative RT-PCR, Titration

    Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).

    Journal: Journal of Virology

    Article Title: Neuralized E3 Ubiquitin Protein Ligase 3 Is an Inducible Antiviral Effector That Inhibits Hepatitis C Virus Assembly by Targeting Viral E1 Glycoprotein

    doi: 10.1128/JVI.01123-18

    Figure Lengend Snippet: Induction of NEURL3 in liver biopsy specimens of HCV-infected patients. The data of NEURL3 mRNA levels in liver biopsy specimens of HCV-infected patients and control patients were obtained from three published studies and analyzed. GEO2R was used to obtain the data, and the t test was used for statistical analysis. (A) NEURL3 expression was analyzed in an mRNA microarray analysis of 87 HCV-associated HCC liver samples and 8 control samples (HCV- and hepatitis B virus [HBV]-free metastatic liver tumors). Raw microarray data were obtained from Gene Expression Omnibus (GSE17856). (B) NEURL3 expression was analyzed in an mRNA microarray analysis of 24 HCV-positive liver biopsy samples from HCV-induced cirrhosis patients and 6 HCV-negative liver biopsy samples from patients with non-HCV-associated liver diseases (autoimmune hepatitis, alcohol-induced cirrhosis, or primary biliary cirrhosis). Raw microarray data were obtained from GEO (GSE15331). (C) NEURL3 expression was analyzed in an RNA-Seq analysis of 28 liver biopsy specimens from chronic hepatitis C and 6 control patients with non-HCV-associated liver diseases. The normalized raw data were obtained from GEO (GSE84346).

    Article Snippet: The primary antibodies included rabbit anti-NEURL3 (Sigma-Aldrich), mouse anti-β-actin (Santa Cruz), mouse anti-Flag (Sigma), mouse anti-E1 monoclonal antibodies A4 (kindly provided by Jean Dubuisson) ( 37 ); mouse anti-E2 and mouse anti-core antibodies were generated by our laboratory, and goat-anti mouse horseradish peroxidase (HRP) antibody and goat-anti rabbit HRP antibody were obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Expressing, Microarray, RNA Sequencing Assay