phospho irf3 ser396  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho irf3 ser396
    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
    Phospho Irf3 Ser396, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells"

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S160645

    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
    Figure Legend Snippet: DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.

    Techniques Used: Western Blot, Binding Assay

    DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.
    Figure Legend Snippet: DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.

    Techniques Used: Binding Assay

    DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.
    Figure Legend Snippet: DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.

    Techniques Used: Western Blot

    A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.
    Figure Legend Snippet: A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.

    Techniques Used: Inhibition, Binding Assay

    rabbit monoclonal anti phospho irf 3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho irf 3
    Rabbit Monoclonal Anti Phospho Irf 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    irf 3 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc irf 3 rabbit mab
    Irf 3 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho irf 3 ser396 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho irf 3 ser396 rabbit mab
    Phospho Irf 3 Ser396 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho irf3 ser396  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho irf3 ser396
    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
    Phospho Irf3 Ser396, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho irf3 ser396/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho irf3 ser396 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells"

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S160645

    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
    Figure Legend Snippet: DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.

    Techniques Used: Western Blot, Binding Assay

    DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.
    Figure Legend Snippet: DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.

    Techniques Used: Binding Assay

    DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.
    Figure Legend Snippet: DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.

    Techniques Used: Western Blot

    A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.
    Figure Legend Snippet: A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.

    Techniques Used: Inhibition, Binding Assay

    phospho irf3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho irf3
    The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of <t>IRF3</t> and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).
    Phospho Irf3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2"

    Article Title: N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2

    Journal: Autophagy

    doi: 10.1080/15548627.2017.1345411

    The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).
    Figure Legend Snippet: The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).

    Techniques Used: Expressing, Quantitative RT-PCR, One-tailed Test, Incubation

    The DHA-mediated inflammatory tolerance is related to the formation of SQSTM1/p62-bodies. (A) IB analysis of MDMs treated with DHA (70 μM) for the indicated times followed by LPS for 1 h. The quantification (below) represents mean fold changes ± SEM of DHA + LPS compared to vehicle + LPS on a log scale, n = 6, (Friedman test with Dunn's). (B) QRT-PCR analysis of CXCL10 levels in MDM after 16 h DHA with and without addition of 5 mM NAC followed by 3 h LPS, n = 3, (repeated-measures ANOVA with Dunnett's). (C) QRT-PCR analysis of CXCL10 in SQSTM1 siRNA-transfected MDMs treated with DHA for 16 h and LPS for 3 h, n = 5, (Friedman test with Dunn's). (D) IB analysis of IRF3, RELA and IKBKB phosphorylation in control and SQSTM1 knockdown MDMs, n = 6 (Friedman test with Dunn's). (E) CXCL10 protein levels in Sqstm1 +/+ (n = 8) and sqstm1 − / − (n = 10) male mice were measured by ELISA. Serum was analyzed at baseline and at 3 and 6 h after challenge with 3.5 µg/g LPS. ND, not detected.
    Figure Legend Snippet: The DHA-mediated inflammatory tolerance is related to the formation of SQSTM1/p62-bodies. (A) IB analysis of MDMs treated with DHA (70 μM) for the indicated times followed by LPS for 1 h. The quantification (below) represents mean fold changes ± SEM of DHA + LPS compared to vehicle + LPS on a log scale, n = 6, (Friedman test with Dunn's). (B) QRT-PCR analysis of CXCL10 levels in MDM after 16 h DHA with and without addition of 5 mM NAC followed by 3 h LPS, n = 3, (repeated-measures ANOVA with Dunnett's). (C) QRT-PCR analysis of CXCL10 in SQSTM1 siRNA-transfected MDMs treated with DHA for 16 h and LPS for 3 h, n = 5, (Friedman test with Dunn's). (D) IB analysis of IRF3, RELA and IKBKB phosphorylation in control and SQSTM1 knockdown MDMs, n = 6 (Friedman test with Dunn's). (E) CXCL10 protein levels in Sqstm1 +/+ (n = 8) and sqstm1 − / − (n = 10) male mice were measured by ELISA. Serum was analyzed at baseline and at 3 and 6 h after challenge with 3.5 µg/g LPS. ND, not detected.

    Techniques Used: Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

    irf3  (Cell Signaling Technology Inc)


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    anti phospho irf3 ser396  (Cell Signaling Technology Inc)


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    rabbit irf 3 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit irf 3 antibody
    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 <t>(IRF-3)</t> and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
    Rabbit Irf 3 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes"

    Article Title: Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

    Journal: Gut

    doi: 10.1136/gut.2007.147264

    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
    Figure Legend Snippet: NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Techniques Used: Expressing, Translocation Assay, In Vitro, Polyacrylamide Gel Electrophoresis, SDS Page, Transfection, Western Blot, Functional Assay, Mutagenesis, Sequencing, Negative Control

    irf 3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc irf 3
    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 <t>(IRF-3)</t> and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
    Irf 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes"

    Article Title: Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

    Journal: Gut

    doi: 10.1136/gut.2007.147264

    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
    Figure Legend Snippet: NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Techniques Used: Expressing, Translocation Assay, In Vitro, Polyacrylamide Gel Electrophoresis, SDS Page, Transfection, Western Blot, Functional Assay, Mutagenesis, Sequencing, Negative Control

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    Cell Signaling Technology Inc phospho irf3 ser396
    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
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    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
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    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
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    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and <t>TBK1/IRF3</t> pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.
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    The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of <t>IRF3</t> and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).
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    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 <t>(IRF-3)</t> and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
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    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 <t>(IRF-3)</t> and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.
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    Image Search Results


    DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.

    Journal: Drug Design, Development and Therapy

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    doi: 10.2147/DDDT.S160645

    Figure Lengend Snippet: DCEO affected the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO at the indicated concentrations for 1 h and then stimulated with LPS for 30 or 60 min. Total and phosphorylated forms of individual proteins were detected by Western blot ( A and B ). A representative of three independent experiments is shown. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p38, p38 mitogen-activated protein kinase; IKKα/β, IκB kinase α/β; TAK1, TGF β-activated kinase 1; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase.

    Article Snippet: Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IRF3, phospho-IRF3 (Ser396), IκBα, phospho-IκBα (Ser32), IκB kinase α/β (IKKα/β), phospho-IKKα/β (Ser176/180), extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr180/Tyr182), phosphoinositide (PI) 3-kinase p85, phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), TGF β-activated kinase 1 (TAK1), phospho-TAK1 (Ser412), interleukin-1 receptor- associated kinase 1 (IRAK1), IRAK4, TANK-binding kinase 1 (TBK1), phospho-TBK1 (Ser172), c-Fos, phospho-c-Fos (Ser32), and goat anti-mouse (#4408S) conjugated to Alexa Fluor 488 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Western Blot, Binding Assay

    DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.

    Journal: Drug Design, Development and Therapy

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    doi: 10.2147/DDDT.S160645

    Figure Lengend Snippet: DCEO modulates the molecular components of the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in LPS-stimulated RAW264.7 cells. Notes: Bar graphs show the ratio of ( A ) IRAK1/GAPDH, p-TAK1/TAK1, p-IKKα/β/IKKα/β, p-TBK1/TBK1, IRAK4/GAPDH, p-p38/p38, p-ERK/ERK, and p-JNK/JNK In RAW264.7 cells. ( B ) p-PI3K/PI3K, p-Akt/Akt, p-IκB/IκB, p-p65/p65, p-c-Jun/c-Jun, p-IRF3/IRF3, and p-c-Fos/c-Fos in RAW264.7 cells. Data are the mean±SEM of three independent experiments. * P <0.05 and ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test. Abbreviations: DCEO, Dingchuan tang essential oil; IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor- kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; p, phosphorylated; TAK1, TGF β-activated kinase 1; IKKα/β, IκB kinase α/β; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; p38, p38 mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SEM, standard error of the mean.

    Article Snippet: Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IRF3, phospho-IRF3 (Ser396), IκBα, phospho-IκBα (Ser32), IκB kinase α/β (IKKα/β), phospho-IKKα/β (Ser176/180), extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr180/Tyr182), phosphoinositide (PI) 3-kinase p85, phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), TGF β-activated kinase 1 (TAK1), phospho-TAK1 (Ser412), interleukin-1 receptor- associated kinase 1 (IRAK1), IRAK4, TANK-binding kinase 1 (TBK1), phospho-TBK1 (Ser172), c-Fos, phospho-c-Fos (Ser32), and goat anti-mouse (#4408S) conjugated to Alexa Fluor 488 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Binding Assay

    DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.

    Journal: Drug Design, Development and Therapy

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    doi: 10.2147/DDDT.S160645

    Figure Lengend Snippet: DCEO inhibited nuclear localization of NF-κB, AP-1, and IRF3 in LPS-stimulated RAW264.7 cells. Notes: Cells were treated with DCEO with indicated concentrations for 1 h and then stimulated with LPS for 60 min. The cytoplasmic and nuclear protein levels of NF-κB (p65 and p50), c-Jun, and IRF3 were determined by Western blot ( A ). Bar graphs show the relative levels of cytoplasmic and nuclear p65, p50, IRF3, and c-Jun. Data are the mean SEM of three independent experiments. ** P <0.01 vs unstimulated cells. *** P <0.05 and **** P <0.01 vs LPS-stimulated cells determined by Dunnett’s multiple comparisons test ( B ). Abbreviations: DCEO, Dingchuan tang essential oil; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; IRF3, interferon regulatory factor 3; LPS, lipopolysaccharide; SEM, standard error of the mean.

    Article Snippet: Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IRF3, phospho-IRF3 (Ser396), IκBα, phospho-IκBα (Ser32), IκB kinase α/β (IKKα/β), phospho-IKKα/β (Ser176/180), extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr180/Tyr182), phosphoinositide (PI) 3-kinase p85, phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), TGF β-activated kinase 1 (TAK1), phospho-TAK1 (Ser412), interleukin-1 receptor- associated kinase 1 (IRAK1), IRAK4, TANK-binding kinase 1 (TBK1), phospho-TBK1 (Ser172), c-Fos, phospho-c-Fos (Ser32), and goat anti-mouse (#4408S) conjugated to Alexa Fluor 488 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Western Blot

    A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.

    Journal: Drug Design, Development and Therapy

    Article Title: Dingchuan tang essential oil inhibits the production of inflammatory mediators via suppressing the IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 pathways in lipopolysaccharide-stimulated RAW264.7 cells

    doi: 10.2147/DDDT.S160645

    Figure Lengend Snippet: A schematic diagram showing the association of IRAK/NF-κB, IRAK/AP-1, and TBK1/IRF3 signaling inhibition with the inhibitory effects of DCEO on inflammatory mediators. Abbreviations: IRAK, interleukin-1 receptor-associated kinase; NF-κB, nuclear factor-kappa B; AP-1, activator protein-1; TBK1, TANK-binding kinase 1; IRF3, interferon regulatory factor 3; DCEO, Dingchuan tang essential oil; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; CCL-5, chemokine (C-C motif) ligand 5; IKKα/β, IκB kinase α/β; Akt, protein kinase B; PI3K, phosphatidylinositol 3-kinase; TAK1, TGF β-activated kinase 1; MyD88, myeloid differentiation factor 88; TLR4, toll-like receptor 4; LPS, lipopolysaccharide.

    Article Snippet: Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IRF3, phospho-IRF3 (Ser396), IκBα, phospho-IκBα (Ser32), IκB kinase α/β (IKKα/β), phospho-IKKα/β (Ser176/180), extracellular signal-regulated kinase (ERK), phospho-ERK (Thr202/Tyr204), c-Jun N-terminal kinase (JNK), phospho-JNK (Thr183/Tyr185), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr180/Tyr182), phosphoinositide (PI) 3-kinase p85, phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199), TGF β-activated kinase 1 (TAK1), phospho-TAK1 (Ser412), interleukin-1 receptor- associated kinase 1 (IRAK1), IRAK4, TANK-binding kinase 1 (TBK1), phospho-TBK1 (Ser172), c-Fos, phospho-c-Fos (Ser32), and goat anti-mouse (#4408S) conjugated to Alexa Fluor 488 antibodies were obtained from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Inhibition, Binding Assay

    The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).

    Journal: Autophagy

    Article Title: N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2

    doi: 10.1080/15548627.2017.1345411

    Figure Lengend Snippet: The n-3 PUFA DHA strongly reduced expression of CXCL10 by affecting several signaling pathways. (A) MDMs were treated with DHA (70 µM) for 16 h before stimulation with LPS (100 ng/ml) for 3 h. Transcript levels of 579 genes (nCounter GX Human Immunology kit assay) were determined. Transcripts that were more than 1.5-times increased by LPS in both donors were selected and the fold change between vehicle- and DHA-treated cells calculated, mean ± SD, n = 2. (B) QRT-PCR analysis of CXCL10 and (C) TNF in MDM treated with DHA, OA or AA for 16 h and LPS for 3 h, n = 5 (Friedman test with Dunn's). (D) IB analysis and quantification of IRF3 and RELA phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 h, n = 5. (Friedman test with Dunn's), of (E) TBK1, IKBKB, STAT1 and STAT3 phosphorylation in MDMs treated with DHA, OA or AA for 16 h and LPS for 1 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test, one-tailed) and of (F) IRF1 and RELA in cytosolic (Cyt) and nuclear (Nuc) fractions in MDMs treated with DHA, OA or AA for 16 h and LPS for 2 or 4 h, n = 5 (Wilcoxon matched-pairs signed rank test). (G) IFNB1 mRNA levels after incubation with DHA for 16 h and LPS for 2 h, n = 6 (Wilcoxon matched-pairs signed rank test).

    Article Snippet: The following were from Cell Signaling Technology: IKBKB/IKKβ (8943); IRF1 (4302); IRF3 (4302S), MAP1LC3B (3868); MAPK1/3/p44/42 (9107); RELA/p65 (3034); phospho-CHUK/IKBKB/IKKα/β (S176/180; 2697); phospho-IRF3 (S396; 4302); phospho-NFKB2/p105 (S933; 4806); phospho-RELA/p65 (S536; 3033); phospho-STAT1 (Y701; 7649); phospho-STAT3 (Y705; 9145); phospho-TBK1 (S172; 5483); STAT1 (14994); STAT3 (9139); TAX1BP1 (5105); TBK1 (3013).

    Techniques: Expressing, Quantitative RT-PCR, One-tailed Test, Incubation

    The DHA-mediated inflammatory tolerance is related to the formation of SQSTM1/p62-bodies. (A) IB analysis of MDMs treated with DHA (70 μM) for the indicated times followed by LPS for 1 h. The quantification (below) represents mean fold changes ± SEM of DHA + LPS compared to vehicle + LPS on a log scale, n = 6, (Friedman test with Dunn's). (B) QRT-PCR analysis of CXCL10 levels in MDM after 16 h DHA with and without addition of 5 mM NAC followed by 3 h LPS, n = 3, (repeated-measures ANOVA with Dunnett's). (C) QRT-PCR analysis of CXCL10 in SQSTM1 siRNA-transfected MDMs treated with DHA for 16 h and LPS for 3 h, n = 5, (Friedman test with Dunn's). (D) IB analysis of IRF3, RELA and IKBKB phosphorylation in control and SQSTM1 knockdown MDMs, n = 6 (Friedman test with Dunn's). (E) CXCL10 protein levels in Sqstm1 +/+ (n = 8) and sqstm1 − / − (n = 10) male mice were measured by ELISA. Serum was analyzed at baseline and at 3 and 6 h after challenge with 3.5 µg/g LPS. ND, not detected.

    Journal: Autophagy

    Article Title: N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2

    doi: 10.1080/15548627.2017.1345411

    Figure Lengend Snippet: The DHA-mediated inflammatory tolerance is related to the formation of SQSTM1/p62-bodies. (A) IB analysis of MDMs treated with DHA (70 μM) for the indicated times followed by LPS for 1 h. The quantification (below) represents mean fold changes ± SEM of DHA + LPS compared to vehicle + LPS on a log scale, n = 6, (Friedman test with Dunn's). (B) QRT-PCR analysis of CXCL10 levels in MDM after 16 h DHA with and without addition of 5 mM NAC followed by 3 h LPS, n = 3, (repeated-measures ANOVA with Dunnett's). (C) QRT-PCR analysis of CXCL10 in SQSTM1 siRNA-transfected MDMs treated with DHA for 16 h and LPS for 3 h, n = 5, (Friedman test with Dunn's). (D) IB analysis of IRF3, RELA and IKBKB phosphorylation in control and SQSTM1 knockdown MDMs, n = 6 (Friedman test with Dunn's). (E) CXCL10 protein levels in Sqstm1 +/+ (n = 8) and sqstm1 − / − (n = 10) male mice were measured by ELISA. Serum was analyzed at baseline and at 3 and 6 h after challenge with 3.5 µg/g LPS. ND, not detected.

    Article Snippet: The following were from Cell Signaling Technology: IKBKB/IKKβ (8943); IRF1 (4302); IRF3 (4302S), MAP1LC3B (3868); MAPK1/3/p44/42 (9107); RELA/p65 (3034); phospho-CHUK/IKBKB/IKKα/β (S176/180; 2697); phospho-IRF3 (S396; 4302); phospho-NFKB2/p105 (S933; 4806); phospho-RELA/p65 (S536; 3033); phospho-STAT1 (Y701; 7649); phospho-STAT3 (Y705; 9145); phospho-TBK1 (S172; 5483); STAT1 (14994); STAT3 (9139); TAX1BP1 (5105); TBK1 (3013).

    Techniques: Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay

    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Journal: Gut

    Article Title: Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

    doi: 10.1136/gut.2007.147264

    Figure Lengend Snippet: NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Article Snippet: Proteins were transferred to an Invitrolon/polyvinylidene difluoride (PVDF) membrane (Invitrogen) for 1 h at 30 V. For detection of IRF-3, NF-κB and IPS-1, a rabbit-IRF-3 antibody (#4962; Cell Signaling, Danvers, Massachusetts, USA), a rabbit-NF-κB p65 antibody (#3034; Cell Signaling) or a rabbit-IPS-1 antibody (AT107; Alexis Biochemicals, San Diego, California, USA) was used at a dilution of 1:1000, 1:1000 and 1:2000, respectively.

    Techniques: Expressing, Translocation Assay, In Vitro, Polyacrylamide Gel Electrophoresis, SDS Page, Transfection, Western Blot, Functional Assay, Mutagenesis, Sequencing, Negative Control

    NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Journal: Gut

    Article Title: Cleavage of the IPS-1/Cardif/MAVS/VISA does not inhibit T cell-mediated elimination of hepatitis C virus non-structural 3/4A-expressing hepatocytes

    doi: 10.1136/gut.2007.147264

    Figure Lengend Snippet: NS3/4A-protein expression in mice disturbs responses to synthetic dsRNA. The nuclear translocation of interferon regulatory factor 3 (IRF-3) and nuclear factor κB (NF-κB) by intravenous poly(I:C) is reduced in stable NS3/4A-Tg mice (A), but not by tumour necrosis factor α (TNFα)/ d- GalN treatment (B), representing a retinoic acid inducible gene-I (RIG-I)-independent pathway. Cleavage of murine (m) interferon β (IFNβ) promoter stimulator-1 (IPS-1) by the NS3/4A protease was determined using a standard in vitro transcription and translation assay in presence of 35 S-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (C). The loading of each lane is indicated under the gel. Expression of mIPS-1 alone generates a single protein band, whereas mIPS-1 co-expressed with wtNS3/4A or coNS3/4A, but not wtNS3 (data not shown), generates two distinct protein bands, one representing mIPS-1 and the other the truncated mIPS-1 (C). Detection of cleaved hIPS-1 (ΔhIPS-1) and ΔmIPS-1 in HepG2 cells (D) co-transfected with human (upper gel) or mouse (lower gel) IPS-1 by HCV NS3/4A and appropriate controls. Uncleaved IPS-1 generates one single band in the western blot analysis. In the presence of a functional NS3/4A protease (wtNS3/4A and coNS3/4A) a cleavage of IPS-1 generates two distinct bands on the membrane. mNS3/4A has a mutation that prevents the release of NS4A and the generation of the complete NS3/4A complex (D). Comparison of the efficiency of human IPS-1 cleavage by the NS3/4A sequence with alanine mutations at residues 111, 112 or 113 of the NS3 protease with the cleavage of murine IPS-1 (E). Also shown is a parallel analysis of the cleavage of mouse IPS-1 by HBcAg (negative control), wild-type NS3, mutant NS3/4A with an impaired protease, and coNS3/4A with a functional protease. All experiments shown have been repeated two to five times.

    Article Snippet: Proteins were transferred to an Invitrolon/polyvinylidene difluoride (PVDF) membrane (Invitrogen) for 1 h at 30 V. For detection of IRF-3, NF-κB and IPS-1, a rabbit-IRF-3 antibody (#4962; Cell Signaling, Danvers, Massachusetts, USA), a rabbit-NF-κB p65 antibody (#3034; Cell Signaling) or a rabbit-IPS-1 antibody (AT107; Alexis Biochemicals, San Diego, California, USA) was used at a dilution of 1:1000, 1:1000 and 1:2000, respectively.

    Techniques: Expressing, Translocation Assay, In Vitro, Polyacrylamide Gel Electrophoresis, SDS Page, Transfection, Western Blot, Functional Assay, Mutagenesis, Sequencing, Negative Control