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phosphorylated iκbα (af1870) antibodies  (Beyotime)


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    Beyotime phosphorylated iκbα (af1870) antibodies
    H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, <t>IRF3,</t> and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.
    Phosphorylated Iκbα (Af1870) Antibodies, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphorylated iκbα (af1870) antibodies/product/Beyotime
    Average 90 stars, based on 1 article reviews
    phosphorylated iκbα (af1870) antibodies - by Bioz Stars, 2026-02
    90/100 stars

    Images

    1) Product Images from "The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice"

    Article Title: The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice

    Journal: Journal of Virology

    doi: 10.1128/JVI.01640-20

    H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, IRF3, and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, IRF3, and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.

    Techniques Used: Luciferase, Plasmid Preparation, Control, Transfection, Infection, Phospho-proteomics, SDS Page, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay

    Recombinant PR8-H7N9NP influenza virus induces IFN expression and inhibits viral replication in A549 cells. (A) rPR8-H7N9NP elevates the transcriptional expression of antiviral genes. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 h and 24 h before qPCR analysis. (B) rPR8-H7N9NP potentiates IFN-β secretion. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 and 24 h before analysis of IFN-β secretion by ELISA. (C) rPR8-H7N9NP enhances the phosphorylation of TBK1, IRF3, and IκBα. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to the GAPDH control. (D) Growth kinetics of rPR8 and rPR8-H7N9NP viruses in A549 cells. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP virus (MOI = 0.1). Virus titers were determined in culture supernatants collected at the indicated time points. (E) Polymerase activities of PR8 NP and H7N9 NP. A minigenome assay was conducted in HEK293 cells transfected with reporter plasmids pPolI-Luc (25 ng) and pRL-TK (10 ng) and pCAGGS plasmid constructs expressing the PR8 H1N1 PB2, PB1, and PA genes, with the PR8 H1N1 NP or H7N9 NP gene (25 ng each). Cell extracts were harvested 24 h posttransfection, and luciferase activity was assayed by using the luciferase assay system. The assay was standardized against the Renilla luciferase activity. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: Recombinant PR8-H7N9NP influenza virus induces IFN expression and inhibits viral replication in A549 cells. (A) rPR8-H7N9NP elevates the transcriptional expression of antiviral genes. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 h and 24 h before qPCR analysis. (B) rPR8-H7N9NP potentiates IFN-β secretion. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 and 24 h before analysis of IFN-β secretion by ELISA. (C) rPR8-H7N9NP enhances the phosphorylation of TBK1, IRF3, and IκBα. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to the GAPDH control. (D) Growth kinetics of rPR8 and rPR8-H7N9NP viruses in A549 cells. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP virus (MOI = 0.1). Virus titers were determined in culture supernatants collected at the indicated time points. (E) Polymerase activities of PR8 NP and H7N9 NP. A minigenome assay was conducted in HEK293 cells transfected with reporter plasmids pPolI-Luc (25 ng) and pRL-TK (10 ng) and pCAGGS plasmid constructs expressing the PR8 H1N1 PB2, PB1, and PA genes, with the PR8 H1N1 NP or H7N9 NP gene (25 ng each). Cell extracts were harvested 24 h posttransfection, and luciferase activity was assayed by using the luciferase assay system. The assay was standardized against the Renilla luciferase activity. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Recombinant, Virus, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay

    H7N9 NP specifically associates with TRAF3. (A) Overexpressed NP interacts with TRAF3. HEK293 cells (1 × 106) were transfected with plasmids encoding Flag-NP (500 ng) and HA-tagged RLR-mediated IFN signaling molecules (RIG-I [700 ng], MAVS [100 ng], TRAF3 [200 ng], TRAF6 [200 ng], TBK1 [700 ng], IKKε [1,000 ng], and IRF3 [200 ng]), followed by IP with anti-Flag beads or IgG control and immunoblot analysis with anti-HA. (B) NP interacts with TRAF3 directly. Purified GST-H7N9NP was used to pull down transiently expressed Flag-MAVS, TRAF3, or TRAF6 and then analyzed by immunoblotting with the indicated antibodies. (C) NP interacts with TRAF3 physiologically. A549 cells (1 × 107) were infected with H7N9 virus (MOI = 1) for 24 h, followed by IP with an NP antibody and immunoblot analysis with a TRAF3 antibody, controlled with immunoblot analysis of whole-cell lysates. (D) NP colocalizes with TRAF3 in cell cytoplasm. A549 cells (5 × 105) were transfected with Flag-TRAF3 (200 ng) or Flag-MAVS (100 ng) plasmid for 24 h before infection with H7N9 (MOI = 0.1) for 24 h and then stained with a Flag antibody or an NP primary antibody and Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (green) or Alexa Fluor 594-conjugated anti-rabbit IgG secondary antibody (red). The nucleus was stained by DAPI. Scale bars, 10 μm. (E) The domain of TRAF3 from aa 282 to 347 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 108 [1,500 ng], aa 1 to 281 [1,500 ng], aa 1 to 347 [1,500 ng], aa 109 to 568 [1,500 ng], aa 282 to 568 [1,500 ng], and aa 348 to 568 [1,500 ng]). (F) The domain of NP from aa 181 to 370 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 180 [1,000 ng], aa 1 to 370 [500 ng], aa 181 to 370 [1,000 ng], aa 181 to 498 [700 ng], and aa 371 to 498 [1,500 ng]).
    Figure Legend Snippet: H7N9 NP specifically associates with TRAF3. (A) Overexpressed NP interacts with TRAF3. HEK293 cells (1 × 106) were transfected with plasmids encoding Flag-NP (500 ng) and HA-tagged RLR-mediated IFN signaling molecules (RIG-I [700 ng], MAVS [100 ng], TRAF3 [200 ng], TRAF6 [200 ng], TBK1 [700 ng], IKKε [1,000 ng], and IRF3 [200 ng]), followed by IP with anti-Flag beads or IgG control and immunoblot analysis with anti-HA. (B) NP interacts with TRAF3 directly. Purified GST-H7N9NP was used to pull down transiently expressed Flag-MAVS, TRAF3, or TRAF6 and then analyzed by immunoblotting with the indicated antibodies. (C) NP interacts with TRAF3 physiologically. A549 cells (1 × 107) were infected with H7N9 virus (MOI = 1) for 24 h, followed by IP with an NP antibody and immunoblot analysis with a TRAF3 antibody, controlled with immunoblot analysis of whole-cell lysates. (D) NP colocalizes with TRAF3 in cell cytoplasm. A549 cells (5 × 105) were transfected with Flag-TRAF3 (200 ng) or Flag-MAVS (100 ng) plasmid for 24 h before infection with H7N9 (MOI = 0.1) for 24 h and then stained with a Flag antibody or an NP primary antibody and Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (green) or Alexa Fluor 594-conjugated anti-rabbit IgG secondary antibody (red). The nucleus was stained by DAPI. Scale bars, 10 μm. (E) The domain of TRAF3 from aa 282 to 347 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 108 [1,500 ng], aa 1 to 281 [1,500 ng], aa 1 to 347 [1,500 ng], aa 109 to 568 [1,500 ng], aa 282 to 568 [1,500 ng], and aa 348 to 568 [1,500 ng]). (F) The domain of NP from aa 181 to 370 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 180 [1,000 ng], aa 1 to 370 [500 ng], aa 181 to 370 [1,000 ng], aa 181 to 498 [700 ng], and aa 371 to 498 [1,500 ng]).

    Techniques Used: Transfection, Control, Western Blot, Purification, Infection, Virus, Plasmid Preparation, Staining, Co-Immunoprecipitation Assay



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    phosphorylated iκbα (af1870) antibodies  (Beyotime)
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    H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, <t>IRF3,</t> and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.
    Phosphorylated Iκbα (Af1870) Antibodies, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphorylated iκbα (af1870) antibodies/product/Beyotime
    Average 90 stars, based on 1 article reviews
    phosphorylated iκbα (af1870) antibodies - by Bioz Stars, 2026-02
    90/100 stars
    		  Buy from Supplier
    phosphorylated iκbα (af1870)  (Beyotime)
    90
    Beyotime phosphorylated iκbα (af1870)
    H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, <t>IRF3,</t> and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.
    Phosphorylated Iκbα (Af1870), supplied by Beyotime, 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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    Average 90 stars, based on 1 article reviews
    phosphorylated iκbα (af1870) - by Bioz Stars, 2026-02
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    		  Buy from Supplier

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    H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, IRF3, and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice

    doi: 10.1128/JVI.01640-20

    Figure Lengend Snippet: H7N9 NP positively regulates type I IFN signaling. (A to C) NP promotes IFN-related promoter activities. Luciferase reporter plasmid (IFN-β-Luc [100 ng], ISRE-Luc [100 ng], or NF-κB-Luc [50 ng]) and pRL-TK (10 ng) plasmid were cotransfected into HEK293 cells (1 × 105), along with a vector control plasmid or pRK-NP (200 ng), and induced by TNF-α (50 ng/ml) treatment, poly(I·C) (10 μg/ml) transfection, or SeV (MOI = 1) infection. Cell lysates were used for luciferase assays after transfection and infection. (D) NP upregulates transcription of antiviral genes. NP-overexpressing A549 cells (1 × 106) were infected with H7N9 (MOI = 1) or SeV (MOI = 1) or transfected with RIG-I-CARD (RIG-IN) (1200 ng)/poly(I·C) (10 μg/ml) for 24 h before qPCR analysis. (E) H7N9 NP enhances the phosphorylation of TBK1, IRF3, and IκBα. After HEK293 cells (2 × 106) were transfected with a vector control (500 ng) or NP (500 ng) plasmid for 24 h, the cells were infected with SeV (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to a GAPDH control. (F) Overexpression of NP promotes RIG-IN/SeV/H7N9-induced IFN-β secretion. NP-overexpressing A549 cells (2 × 106) were transfected with RIG-IN (1200 ng) or infected with SeV (MOI = 1) or H7N9 (MOI = 1) for 24 h before being analyzed for IFN-β secretion by ELISA. **, P < 0.01; ***, P < 0.001.

    Article Snippet: Phosphorylated IκBα (AF1870) and IRF3 (AF1594) antibodies, 4′,6-diamidino-2-phenylindole (DAPI; C1005), and NP-40 (ST366) were from Beyotime (Shanghai, China).

    Techniques: Luciferase, Plasmid Preparation, Control, Transfection, Infection, Phospho-proteomics, SDS Page, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay

    Recombinant PR8-H7N9NP influenza virus induces IFN expression and inhibits viral replication in A549 cells. (A) rPR8-H7N9NP elevates the transcriptional expression of antiviral genes. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 h and 24 h before qPCR analysis. (B) rPR8-H7N9NP potentiates IFN-β secretion. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 and 24 h before analysis of IFN-β secretion by ELISA. (C) rPR8-H7N9NP enhances the phosphorylation of TBK1, IRF3, and IκBα. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to the GAPDH control. (D) Growth kinetics of rPR8 and rPR8-H7N9NP viruses in A549 cells. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP virus (MOI = 0.1). Virus titers were determined in culture supernatants collected at the indicated time points. (E) Polymerase activities of PR8 NP and H7N9 NP. A minigenome assay was conducted in HEK293 cells transfected with reporter plasmids pPolI-Luc (25 ng) and pRL-TK (10 ng) and pCAGGS plasmid constructs expressing the PR8 H1N1 PB2, PB1, and PA genes, with the PR8 H1N1 NP or H7N9 NP gene (25 ng each). Cell extracts were harvested 24 h posttransfection, and luciferase activity was assayed by using the luciferase assay system. The assay was standardized against the Renilla luciferase activity. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Journal of Virology

    Article Title: The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice

    doi: 10.1128/JVI.01640-20

    Figure Lengend Snippet: Recombinant PR8-H7N9NP influenza virus induces IFN expression and inhibits viral replication in A549 cells. (A) rPR8-H7N9NP elevates the transcriptional expression of antiviral genes. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 h and 24 h before qPCR analysis. (B) rPR8-H7N9NP potentiates IFN-β secretion. A549 cells (1 × 106) were infected with rPR8-H7N9NP or rPR8 (MOI = 1) for 12 and 24 h before analysis of IFN-β secretion by ELISA. (C) rPR8-H7N9NP enhances the phosphorylation of TBK1, IRF3, and IκBα. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP (MOI = 1) for the indicated times. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The number below the corresponding band shows a statistical analysis of the relative density of the indicated molecules normalized to the GAPDH control. (D) Growth kinetics of rPR8 and rPR8-H7N9NP viruses in A549 cells. A549 cells (2 × 106) were infected with rPR8 or rPR8-H7N9NP virus (MOI = 0.1). Virus titers were determined in culture supernatants collected at the indicated time points. (E) Polymerase activities of PR8 NP and H7N9 NP. A minigenome assay was conducted in HEK293 cells transfected with reporter plasmids pPolI-Luc (25 ng) and pRL-TK (10 ng) and pCAGGS plasmid constructs expressing the PR8 H1N1 PB2, PB1, and PA genes, with the PR8 H1N1 NP or H7N9 NP gene (25 ng each). Cell extracts were harvested 24 h posttransfection, and luciferase activity was assayed by using the luciferase assay system. The assay was standardized against the Renilla luciferase activity. Each bar represents the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: Phosphorylated IκBα (AF1870) and IRF3 (AF1594) antibodies, 4′,6-diamidino-2-phenylindole (DAPI; C1005), and NP-40 (ST366) were from Beyotime (Shanghai, China).

    Techniques: Recombinant, Virus, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, SDS Page, Western Blot, Control, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay

    H7N9 NP specifically associates with TRAF3. (A) Overexpressed NP interacts with TRAF3. HEK293 cells (1 × 106) were transfected with plasmids encoding Flag-NP (500 ng) and HA-tagged RLR-mediated IFN signaling molecules (RIG-I [700 ng], MAVS [100 ng], TRAF3 [200 ng], TRAF6 [200 ng], TBK1 [700 ng], IKKε [1,000 ng], and IRF3 [200 ng]), followed by IP with anti-Flag beads or IgG control and immunoblot analysis with anti-HA. (B) NP interacts with TRAF3 directly. Purified GST-H7N9NP was used to pull down transiently expressed Flag-MAVS, TRAF3, or TRAF6 and then analyzed by immunoblotting with the indicated antibodies. (C) NP interacts with TRAF3 physiologically. A549 cells (1 × 107) were infected with H7N9 virus (MOI = 1) for 24 h, followed by IP with an NP antibody and immunoblot analysis with a TRAF3 antibody, controlled with immunoblot analysis of whole-cell lysates. (D) NP colocalizes with TRAF3 in cell cytoplasm. A549 cells (5 × 105) were transfected with Flag-TRAF3 (200 ng) or Flag-MAVS (100 ng) plasmid for 24 h before infection with H7N9 (MOI = 0.1) for 24 h and then stained with a Flag antibody or an NP primary antibody and Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (green) or Alexa Fluor 594-conjugated anti-rabbit IgG secondary antibody (red). The nucleus was stained by DAPI. Scale bars, 10 μm. (E) The domain of TRAF3 from aa 282 to 347 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 108 [1,500 ng], aa 1 to 281 [1,500 ng], aa 1 to 347 [1,500 ng], aa 109 to 568 [1,500 ng], aa 282 to 568 [1,500 ng], and aa 348 to 568 [1,500 ng]). (F) The domain of NP from aa 181 to 370 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 180 [1,000 ng], aa 1 to 370 [500 ng], aa 181 to 370 [1,000 ng], aa 181 to 498 [700 ng], and aa 371 to 498 [1,500 ng]).

    Journal: Journal of Virology

    Article Title: The Nucleoprotein of H7N9 Influenza Virus Positively Regulates TRAF3-Mediated Innate Signaling and Attenuates Viral Virulence in Mice

    doi: 10.1128/JVI.01640-20

    Figure Lengend Snippet: H7N9 NP specifically associates with TRAF3. (A) Overexpressed NP interacts with TRAF3. HEK293 cells (1 × 106) were transfected with plasmids encoding Flag-NP (500 ng) and HA-tagged RLR-mediated IFN signaling molecules (RIG-I [700 ng], MAVS [100 ng], TRAF3 [200 ng], TRAF6 [200 ng], TBK1 [700 ng], IKKε [1,000 ng], and IRF3 [200 ng]), followed by IP with anti-Flag beads or IgG control and immunoblot analysis with anti-HA. (B) NP interacts with TRAF3 directly. Purified GST-H7N9NP was used to pull down transiently expressed Flag-MAVS, TRAF3, or TRAF6 and then analyzed by immunoblotting with the indicated antibodies. (C) NP interacts with TRAF3 physiologically. A549 cells (1 × 107) were infected with H7N9 virus (MOI = 1) for 24 h, followed by IP with an NP antibody and immunoblot analysis with a TRAF3 antibody, controlled with immunoblot analysis of whole-cell lysates. (D) NP colocalizes with TRAF3 in cell cytoplasm. A549 cells (5 × 105) were transfected with Flag-TRAF3 (200 ng) or Flag-MAVS (100 ng) plasmid for 24 h before infection with H7N9 (MOI = 0.1) for 24 h and then stained with a Flag antibody or an NP primary antibody and Alexa Fluor 488-conjugated anti-mouse IgG secondary antibody (green) or Alexa Fluor 594-conjugated anti-rabbit IgG secondary antibody (red). The nucleus was stained by DAPI. Scale bars, 10 μm. (E) The domain of TRAF3 from aa 282 to 347 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 108 [1,500 ng], aa 1 to 281 [1,500 ng], aa 1 to 347 [1,500 ng], aa 109 to 568 [1,500 ng], aa 282 to 568 [1,500 ng], and aa 348 to 568 [1,500 ng]). (F) The domain of NP from aa 181 to 370 is required for NP interaction with TRAF3. Shown are co-IP and immunoblot analysis of HEK293 cells (1 × 106) transfected with HA-NP and Flag-TRAF3 with its indicated truncations (aa 1 to 180 [1,000 ng], aa 1 to 370 [500 ng], aa 181 to 370 [1,000 ng], aa 181 to 498 [700 ng], and aa 371 to 498 [1,500 ng]).

    Article Snippet: Phosphorylated IκBα (AF1870) and IRF3 (AF1594) antibodies, 4′,6-diamidino-2-phenylindole (DAPI; C1005), and NP-40 (ST366) were from Beyotime (Shanghai, China).

    Techniques: Transfection, Control, Western Blot, Purification, Infection, Virus, Plasmid Preparation, Staining, Co-Immunoprecipitation Assay