trif Cell Signaling Technology Inc Search Results


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  • 92
    Cell Signaling Technology Inc trif
    <t>MyD88</t> dependent pathway predominates in S. uberis infection. (A, B) Immunohistochemistry was used to analyze the expression of MyD88 and <t>TRIF</t> in mammary glands. Data are presented as the means ± SEM (n= 6). * ( P
    Trif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cell Signaling Technology Inc anti trif
    Virus infection induces FOSL1 expression and cytoplasmic translocation leading to inhibition of K63 ubiquitination of TRAF3. (A) Confocal microscopy analysis of FOSL1 in 293T cells with or without poly(I:C) stimulation; nuclei were detected with DAPI staining (blue). (B) Images of FOSL1 expression in bone marrow-derived macrophage (BMDM) after incubation with infected red blood cells (iRBCs) for 24 h. (C) A schematic diagram showing sequences of NLS/NES (nuclear localization signals/nuclear export signals) (predicted at http://www.psort.org/ ) of FOSL1 mutants. Amino acid substitutions in the NLS and NES motifs are indicated in red. (D) Confocal microscopy images of 293T cells transfected with plasmid containing GFP-tagged FOSL1-WT, GFP-tagged FOSL1-NES, or GFP-tagged FOSL1-NLS, followed by poly(I:C) stimulation or no stimulation (NT) overnight. (E) Luciferase activity in FOSL1 knockout 293T cells that were transfected with ISRE-luc reporter plasmid together with expression plasmids for <t>MAVS,</t> <t>TRIF,</t> or cGAS plus STING (cG/ST) as well as FOSL1 WT or mutants as indicated. (F) Cell lysates from FOSL1 KO 293T cells transfected with plasmids for FLAG-tagged TRAF3 and HA-tagged K63, as well as MYC-tagged FOSL1 or various MYC-tagged FOSL1 mutants, followed by IP with anti-FLAG and Western blot analysis with the antibodies as indicated. (G) Confocal microscopy analysis of FOSL1 and TRAF3 in BMDMs with or without poly(I:C) stimulation. Nuclei were detected with DAPI staining (blue). *, P
    Anti Trif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc rabbit anti trif
    siRNA against <t>TRIF,</t> MDA5, but not RIG-I reduced RV1B-induced <t>IRF3</t> dimerization
    Rabbit Anti Trif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc anti trif s219
    siRNA against <t>TRIF,</t> MDA5, but not RIG-I reduced RV1B-induced <t>IRF3</t> dimerization
    Anti Trif S219, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    R&D Systems anti human trif ticam
    MyD88 is required for inflammasome sensing of HIV and HCV. Knockdowns of the TLR adaptors MyD88 ( A , B ) and <t>TICAM-1</t> <t>(TRIF,</t> E , F ) were generated and confirmed by previously described RNA interference techniques. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which MyD88 was knocked down were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( C , G ) and IL-18 secretion measured at 24 h ( D , H ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in MyD88 ( C , D ) or TICAM-1 ( G , H ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.001 compared to the scramble siRNA transfected cells.
    Anti Human Trif Ticam, supplied by R&D Systems, 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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    Cell Signaling Technology Inc anti trif rabbit polyclonal antibody
    MyD88 is required for inflammasome sensing of HIV and HCV. Knockdowns of the TLR adaptors MyD88 ( A , B ) and <t>TICAM-1</t> <t>(TRIF,</t> E , F ) were generated and confirmed by previously described RNA interference techniques. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which MyD88 was knocked down were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( C , G ) and IL-18 secretion measured at 24 h ( D , H ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in MyD88 ( C , D ) or TICAM-1 ( G , H ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.001 compared to the scramble siRNA transfected cells.
    Anti Trif Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti trif
    Effects of asunaprevir on replication of <t>HCV</t> in JFH-1- infected . Huh 7.5.1 cells after knockdown of MAVS and <t>TRIF</t> by siRNA. JFH-1-infected Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. HCV core protein, MAVS and TRIF were determined by immunoblotting analysis. The HCV core protein levels relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in the figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P
    Anti Trif, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc tir domain containing adapter protein induced ifnb trif rabbit polyclonal
    Effects of asunaprevir on replication of <t>HCV</t> in JFH-1- infected . Huh 7.5.1 cells after knockdown of MAVS and <t>TRIF</t> by siRNA. JFH-1-infected Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. HCV core protein, MAVS and TRIF were determined by immunoblotting analysis. The HCV core protein levels relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in the figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P
    Tir Domain Containing Adapter Protein Induced Ifnb Trif Rabbit Polyclonal, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    MyD88 dependent pathway predominates in S. uberis infection. (A, B) Immunohistochemistry was used to analyze the expression of MyD88 and TRIF in mammary glands. Data are presented as the means ± SEM (n= 6). * ( P

    Journal: bioRxiv

    Article Title: TLR2 signaling pathway combats Streptococcus uberis infection by inducing production of mitochondrial reactive oxygen species

    doi: 10.1101/809186

    Figure Lengend Snippet: MyD88 dependent pathway predominates in S. uberis infection. (A, B) Immunohistochemistry was used to analyze the expression of MyD88 and TRIF in mammary glands. Data are presented as the means ± SEM (n= 6). * ( P

    Article Snippet: Tissue slices were blocked with 5% bovine serum albumin and incubated with antibodies against MyD88, TRAF6, ECSIT and TRIF (Cell Signaling Technology, Danvers, MA, USA), at 4°C in a humidified chamber.

    Techniques: Infection, Immunohistochemistry, Expressing

    Virus infection induces FOSL1 expression and cytoplasmic translocation leading to inhibition of K63 ubiquitination of TRAF3. (A) Confocal microscopy analysis of FOSL1 in 293T cells with or without poly(I:C) stimulation; nuclei were detected with DAPI staining (blue). (B) Images of FOSL1 expression in bone marrow-derived macrophage (BMDM) after incubation with infected red blood cells (iRBCs) for 24 h. (C) A schematic diagram showing sequences of NLS/NES (nuclear localization signals/nuclear export signals) (predicted at http://www.psort.org/ ) of FOSL1 mutants. Amino acid substitutions in the NLS and NES motifs are indicated in red. (D) Confocal microscopy images of 293T cells transfected with plasmid containing GFP-tagged FOSL1-WT, GFP-tagged FOSL1-NES, or GFP-tagged FOSL1-NLS, followed by poly(I:C) stimulation or no stimulation (NT) overnight. (E) Luciferase activity in FOSL1 knockout 293T cells that were transfected with ISRE-luc reporter plasmid together with expression plasmids for MAVS, TRIF, or cGAS plus STING (cG/ST) as well as FOSL1 WT or mutants as indicated. (F) Cell lysates from FOSL1 KO 293T cells transfected with plasmids for FLAG-tagged TRAF3 and HA-tagged K63, as well as MYC-tagged FOSL1 or various MYC-tagged FOSL1 mutants, followed by IP with anti-FLAG and Western blot analysis with the antibodies as indicated. (G) Confocal microscopy analysis of FOSL1 and TRAF3 in BMDMs with or without poly(I:C) stimulation. Nuclei were detected with DAPI staining (blue). *, P

    Journal: mBio

    Article Title: FOSL1 Inhibits Type I Interferon Responses to Malaria and Viral Infections by Blocking TBK1 and TRAF3/TRIF Interactions

    doi: 10.1128/mBio.02161-16

    Figure Lengend Snippet: Virus infection induces FOSL1 expression and cytoplasmic translocation leading to inhibition of K63 ubiquitination of TRAF3. (A) Confocal microscopy analysis of FOSL1 in 293T cells with or without poly(I:C) stimulation; nuclei were detected with DAPI staining (blue). (B) Images of FOSL1 expression in bone marrow-derived macrophage (BMDM) after incubation with infected red blood cells (iRBCs) for 24 h. (C) A schematic diagram showing sequences of NLS/NES (nuclear localization signals/nuclear export signals) (predicted at http://www.psort.org/ ) of FOSL1 mutants. Amino acid substitutions in the NLS and NES motifs are indicated in red. (D) Confocal microscopy images of 293T cells transfected with plasmid containing GFP-tagged FOSL1-WT, GFP-tagged FOSL1-NES, or GFP-tagged FOSL1-NLS, followed by poly(I:C) stimulation or no stimulation (NT) overnight. (E) Luciferase activity in FOSL1 knockout 293T cells that were transfected with ISRE-luc reporter plasmid together with expression plasmids for MAVS, TRIF, or cGAS plus STING (cG/ST) as well as FOSL1 WT or mutants as indicated. (F) Cell lysates from FOSL1 KO 293T cells transfected with plasmids for FLAG-tagged TRAF3 and HA-tagged K63, as well as MYC-tagged FOSL1 or various MYC-tagged FOSL1 mutants, followed by IP with anti-FLAG and Western blot analysis with the antibodies as indicated. (G) Confocal microscopy analysis of FOSL1 and TRAF3 in BMDMs with or without poly(I:C) stimulation. Nuclei were detected with DAPI staining (blue). *, P

    Article Snippet: Anti-MAVS (3393), anti-TRIF (4596S), anti-FOSL1 (5281), anti-TBK1 (3013), and anti-p-IRF3 (4947) were from Cell Signaling Technology, Inc. (Danvers, MA); Anti-K63 (05-1308) and anti-K48 (05-1307) were from EMD Millipore; anti-IRF3 (sc-9082), anti-TRAF3 (sc-6933), anti-FOSL1 (SC-605), and anti-GFP (sc-8334) were from Santa Cruz Biotechnology (Dallas, TX); anti-MYC-horseradish peroxidase (anti-MYC-HRP) (11814150001) was from Roche Applied Science (Indianapolis, IN); and anti-FLAG-HRP (M2) and anti-β-actin (A1978) were from Sigma (St. Louis, MO).

    Techniques: Infection, Expressing, Translocation Assay, Inhibition, Confocal Microscopy, Staining, Derivative Assay, Incubation, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Knock-Out, Western Blot

    FOSL1 disrupts TBK1 and TRAF3/TRIF interactions and ubiquitination. (A) Cell lysates from 293T cells were transfected with expression plasmids for HA-TBK1, F-TRAF3, F-TRAF6, and F-TRIF with or without MYC-FOSL1, followed by immunoprecipitation (IP) with anti-FLAG and immunoblotting (IB) with antibodies against HA, FLAG, and MYC. (B) Cell lysates from 293T WT and FOSL1 knockdown cells were transfected with HA-TBK1, F-TRAF3, F-TRAF6, and F-TRIF, followed by IP with anti-FLAG and IB with antibodies against HA, FLAG, and endogenous FOSL1. (C) Cell lysates from 293T cells were transfected with expression plasmids for F-TRAF3 and different types of HA-ubiquitin (HA-UB) with or without overexpression of MYC-FOSL1. Co-IP for TRAF3 was performed using anti-FLAG breads and IB with antibodies against HA, FLAG, and MYC. (D) Cell lysates from WT and FOSL1 knockout macrophages (generated by the CRISPR/Cas9 technique) infected with VSV for 18 h (or left uninfected as a control) were subjected to coimmunoprecipitation using anti-TRAF3 antibody and detected with the indicated antibodies in Western blotting. Data are representative of three independent experiments with similar results.

    Journal: mBio

    Article Title: FOSL1 Inhibits Type I Interferon Responses to Malaria and Viral Infections by Blocking TBK1 and TRAF3/TRIF Interactions

    doi: 10.1128/mBio.02161-16

    Figure Lengend Snippet: FOSL1 disrupts TBK1 and TRAF3/TRIF interactions and ubiquitination. (A) Cell lysates from 293T cells were transfected with expression plasmids for HA-TBK1, F-TRAF3, F-TRAF6, and F-TRIF with or without MYC-FOSL1, followed by immunoprecipitation (IP) with anti-FLAG and immunoblotting (IB) with antibodies against HA, FLAG, and MYC. (B) Cell lysates from 293T WT and FOSL1 knockdown cells were transfected with HA-TBK1, F-TRAF3, F-TRAF6, and F-TRIF, followed by IP with anti-FLAG and IB with antibodies against HA, FLAG, and endogenous FOSL1. (C) Cell lysates from 293T cells were transfected with expression plasmids for F-TRAF3 and different types of HA-ubiquitin (HA-UB) with or without overexpression of MYC-FOSL1. Co-IP for TRAF3 was performed using anti-FLAG breads and IB with antibodies against HA, FLAG, and MYC. (D) Cell lysates from WT and FOSL1 knockout macrophages (generated by the CRISPR/Cas9 technique) infected with VSV for 18 h (or left uninfected as a control) were subjected to coimmunoprecipitation using anti-TRAF3 antibody and detected with the indicated antibodies in Western blotting. Data are representative of three independent experiments with similar results.

    Article Snippet: Anti-MAVS (3393), anti-TRIF (4596S), anti-FOSL1 (5281), anti-TBK1 (3013), and anti-p-IRF3 (4947) were from Cell Signaling Technology, Inc. (Danvers, MA); Anti-K63 (05-1308) and anti-K48 (05-1307) were from EMD Millipore; anti-IRF3 (sc-9082), anti-TRAF3 (sc-6933), anti-FOSL1 (SC-605), and anti-GFP (sc-8334) were from Santa Cruz Biotechnology (Dallas, TX); anti-MYC-horseradish peroxidase (anti-MYC-HRP) (11814150001) was from Roche Applied Science (Indianapolis, IN); and anti-FLAG-HRP (M2) and anti-β-actin (A1978) were from Sigma (St. Louis, MO).

    Techniques: Transfection, Expressing, Immunoprecipitation, Over Expression, Co-Immunoprecipitation Assay, Knock-Out, Generated, CRISPR, Infection, Western Blot

    FOSL1 negatively regulates IFN-I signaling in different cell types. (A) Western blot analysis of phosphorylated IRF3 in 293T cells with or without overexpression of FOSL1 at different time points after poly(I:C) stimulation. (B) Western blot analysis of phosphorylated IRF3 in whole-cell lysates (WCL) of 293T cells after transfection with plasmids containing indicated genes encoding various adaptors, with or without overexpression of FOSL1. Vect, vector. (C) Luciferase activities in 293T cells transfected with ISRE-luc reporter plasmid together with plasmids expressing RIG-I, MAVS, cGAS plus STING, TRIF, TBK1, IKKi, or IRF3 and increasing amounts (0, 150, and 300 ng) of FOSL1 overexpression plasmid. (D) Western blot analysis of phosphorylated IRF3 in WT and FOSL1 KO plasmacytoid dendritic cells (pDCs) after stimulation with VSV, poly(I:C), parasite RNA (pRNA), or infected RBCs (iRBCs) for 16 h. (E) The same experiments as described for panel D but performed in trophoblast cells. (F) Real-time qPCR analysis of IFN-β mRNA levels in WT and FOSL1 KO pDCs stimulated with VSV, poly(I:C), parasite RNA (pRNA), or infected RBCs (iRBCs) for 16 h. No treatment (NT), control. The FOSL1 KO cells were generated using the LentiCRISPR/Cas9 system as described in Materials and Methods. *, P

    Journal: mBio

    Article Title: FOSL1 Inhibits Type I Interferon Responses to Malaria and Viral Infections by Blocking TBK1 and TRAF3/TRIF Interactions

    doi: 10.1128/mBio.02161-16

    Figure Lengend Snippet: FOSL1 negatively regulates IFN-I signaling in different cell types. (A) Western blot analysis of phosphorylated IRF3 in 293T cells with or without overexpression of FOSL1 at different time points after poly(I:C) stimulation. (B) Western blot analysis of phosphorylated IRF3 in whole-cell lysates (WCL) of 293T cells after transfection with plasmids containing indicated genes encoding various adaptors, with or without overexpression of FOSL1. Vect, vector. (C) Luciferase activities in 293T cells transfected with ISRE-luc reporter plasmid together with plasmids expressing RIG-I, MAVS, cGAS plus STING, TRIF, TBK1, IKKi, or IRF3 and increasing amounts (0, 150, and 300 ng) of FOSL1 overexpression plasmid. (D) Western blot analysis of phosphorylated IRF3 in WT and FOSL1 KO plasmacytoid dendritic cells (pDCs) after stimulation with VSV, poly(I:C), parasite RNA (pRNA), or infected RBCs (iRBCs) for 16 h. (E) The same experiments as described for panel D but performed in trophoblast cells. (F) Real-time qPCR analysis of IFN-β mRNA levels in WT and FOSL1 KO pDCs stimulated with VSV, poly(I:C), parasite RNA (pRNA), or infected RBCs (iRBCs) for 16 h. No treatment (NT), control. The FOSL1 KO cells were generated using the LentiCRISPR/Cas9 system as described in Materials and Methods. *, P

    Article Snippet: Anti-MAVS (3393), anti-TRIF (4596S), anti-FOSL1 (5281), anti-TBK1 (3013), and anti-p-IRF3 (4947) were from Cell Signaling Technology, Inc. (Danvers, MA); Anti-K63 (05-1308) and anti-K48 (05-1307) were from EMD Millipore; anti-IRF3 (sc-9082), anti-TRAF3 (sc-6933), anti-FOSL1 (SC-605), and anti-GFP (sc-8334) were from Santa Cruz Biotechnology (Dallas, TX); anti-MYC-horseradish peroxidase (anti-MYC-HRP) (11814150001) was from Roche Applied Science (Indianapolis, IN); and anti-FLAG-HRP (M2) and anti-β-actin (A1978) were from Sigma (St. Louis, MO).

    Techniques: Western Blot, Over Expression, Transfection, Plasmid Preparation, Luciferase, Expressing, Infection, Real-time Polymerase Chain Reaction, Generated

    FOSL1 inhibits IFN-I response through modification of signaling molecules upstream of TBK1. (A) Immunoassay of 293T cell extracts transfected with plasmids encoding F-RIG-I, F-MAVS, F-TRAF3, F-TBK1, F-IRF3, and F-TRIF as well as HA-FOSL1, followed by immunoprecipitation (IP) with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (B) Immunoassay of 293T cell extracts transfected with plasmids encoding F-TRAF2, F-TRAF3, F-TRAF5, and F-TRAF6 as well as HA-FOSL1, followed by IP with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (C) IP and immunoblot analysis of THP-1 cell extracts treated with poly(I:C) or medium overnight. (D) Real-time qPCR analysis of IFN-β and ISG56 expression in WT, FOSL1 KO, TRAF3 KO, and FOSL1/TRAF3 double-knockout BMDMs after stimulation with poly(I:C) for 16 h. *, P

    Journal: mBio

    Article Title: FOSL1 Inhibits Type I Interferon Responses to Malaria and Viral Infections by Blocking TBK1 and TRAF3/TRIF Interactions

    doi: 10.1128/mBio.02161-16

    Figure Lengend Snippet: FOSL1 inhibits IFN-I response through modification of signaling molecules upstream of TBK1. (A) Immunoassay of 293T cell extracts transfected with plasmids encoding F-RIG-I, F-MAVS, F-TRAF3, F-TBK1, F-IRF3, and F-TRIF as well as HA-FOSL1, followed by immunoprecipitation (IP) with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (B) Immunoassay of 293T cell extracts transfected with plasmids encoding F-TRAF2, F-TRAF3, F-TRAF5, and F-TRAF6 as well as HA-FOSL1, followed by IP with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (C) IP and immunoblot analysis of THP-1 cell extracts treated with poly(I:C) or medium overnight. (D) Real-time qPCR analysis of IFN-β and ISG56 expression in WT, FOSL1 KO, TRAF3 KO, and FOSL1/TRAF3 double-knockout BMDMs after stimulation with poly(I:C) for 16 h. *, P

    Article Snippet: Anti-MAVS (3393), anti-TRIF (4596S), anti-FOSL1 (5281), anti-TBK1 (3013), and anti-p-IRF3 (4947) were from Cell Signaling Technology, Inc. (Danvers, MA); Anti-K63 (05-1308) and anti-K48 (05-1307) were from EMD Millipore; anti-IRF3 (sc-9082), anti-TRAF3 (sc-6933), anti-FOSL1 (SC-605), and anti-GFP (sc-8334) were from Santa Cruz Biotechnology (Dallas, TX); anti-MYC-horseradish peroxidase (anti-MYC-HRP) (11814150001) was from Roche Applied Science (Indianapolis, IN); and anti-FLAG-HRP (M2) and anti-β-actin (A1978) were from Sigma (St. Louis, MO).

    Techniques: Modification, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Double Knockout

    A schematic illustration of the proposed model of how Cdh1 exerts its tumor suppressor role in part by the negative regulation of WWP2 E3 ligase activity to stabilize PTEN and to suppress the oncogenic Akt signaling. In most cancer cells, where Cdh1 is largely APC-free due to relatively high Cdk activity, Cdh1 mainly directly binds and inhibits WWP2, which leads to the accumulation of various WWP2 substrates including PTEN, TRIF and Rpb1. But further phosphorylation of Cdh1 by Cdk and Plk promotes Cdh1 degradation and the loss of Cdh1 eventually leads to PTEN destabilization and subsequent Akt activation to facilitate tumorigenesis. APC, anaphase-promoting complex/cyclosome.

    Journal: Cell Discovery

    Article Title: Cdh1 inhibits WWP2-mediated ubiquitination of PTEN to suppress tumorigenesis in an APC-independent manner

    doi: 10.1038/celldisc.2015.44

    Figure Lengend Snippet: A schematic illustration of the proposed model of how Cdh1 exerts its tumor suppressor role in part by the negative regulation of WWP2 E3 ligase activity to stabilize PTEN and to suppress the oncogenic Akt signaling. In most cancer cells, where Cdh1 is largely APC-free due to relatively high Cdk activity, Cdh1 mainly directly binds and inhibits WWP2, which leads to the accumulation of various WWP2 substrates including PTEN, TRIF and Rpb1. But further phosphorylation of Cdh1 by Cdk and Plk promotes Cdh1 degradation and the loss of Cdh1 eventually leads to PTEN destabilization and subsequent Akt activation to facilitate tumorigenesis. APC, anaphase-promoting complex/cyclosome.

    Article Snippet: Anti-Aurora A antibody (3092), anti-PTEN antibody (9188) anti-NEDD4L antibody (4013), anti-NEDD4 antibody (2740), anti-TRIF antibody (4596), anti-phospho-Ser473-Akt antibody (4051) and anti-pS9-GSK3β antibody (9323) were purchased from Cell Signaling (Danvers, MA, USA).

    Techniques: Activity Assay, Activation Assay

    Silencing TRIF expression reduces TLR5-dependent signaling in NCM460 cells. A ) and the control cells transfected with the scrambled shRNA construct were stimulated with flagellin (100 ng/ml), followed by evaluating NFκB

    Journal: The Journal of Biological Chemistry

    Article Title: TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells *

    doi: 10.1074/jbc.M110.158394

    Figure Lengend Snippet: Silencing TRIF expression reduces TLR5-dependent signaling in NCM460 cells. A ) and the control cells transfected with the scrambled shRNA construct were stimulated with flagellin (100 ng/ml), followed by evaluating NFκB

    Article Snippet: Antibodies against human TRIF, phospho-ERK1/2, phospho-p38, phospho-Akt, phospho-p105 (NFκB), phospho-p65 (NFκB), ERK1/2, Akt, and MEK1/2 were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Expressing, Transfection, shRNA, Construct

    Tyrosine phosphorylation of TLR3 is required for recruitment of TRIF

    Journal: Science signaling

    Article Title: EGFR is Essential for TLR3 Signaling

    doi: 10.1126/scisignal.2002581

    Figure Lengend Snippet: Tyrosine phosphorylation of TLR3 is required for recruitment of TRIF

    Article Snippet: Antibodies against Src, phospho-Tyr416 -Src and LRRK1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively; anti-human TLR3 (ab13915) and anti-mouse TLR3 (ab53424) were from Abcam (Cambridge, MA, USA); anti-human TRIF (4596), anti-EGFR, anti-ErbB2, and anti-phospho-Ser396 IRF-3 were from Cell Signaling (Danvers, MA, USA); anti-mouse TRIF (LS-C749/22434) were from LifeSpanBioSciences (Seattle, WA, USA); antibodies against IRF-3, His, and GST were from Santa Cruz Biotechnology; anti-phosphotyrosine (4G10 platinum) was from Millipore (Billerica, MA, USA); antibodies against P54, P56, P60, and DRBP76 were raised in our laboratory.

    Techniques:

    EGFR is required for activation of IRF-3 and recruitment of TRIF by TLR3

    Journal: Science signaling

    Article Title: EGFR is Essential for TLR3 Signaling

    doi: 10.1126/scisignal.2002581

    Figure Lengend Snippet: EGFR is required for activation of IRF-3 and recruitment of TRIF by TLR3

    Article Snippet: Antibodies against Src, phospho-Tyr416 -Src and LRRK1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively; anti-human TLR3 (ab13915) and anti-mouse TLR3 (ab53424) were from Abcam (Cambridge, MA, USA); anti-human TRIF (4596), anti-EGFR, anti-ErbB2, and anti-phospho-Ser396 IRF-3 were from Cell Signaling (Danvers, MA, USA); anti-mouse TRIF (LS-C749/22434) were from LifeSpanBioSciences (Seattle, WA, USA); antibodies against IRF-3, His, and GST were from Santa Cruz Biotechnology; anti-phosphotyrosine (4G10 platinum) was from Millipore (Billerica, MA, USA); antibodies against P54, P56, P60, and DRBP76 were raised in our laboratory.

    Techniques: Activation Assay

    EGFR interaction with TLR3 is independent of TLR3 tyrosine residues and TRIF

    Journal: Science signaling

    Article Title: EGFR is Essential for TLR3 Signaling

    doi: 10.1126/scisignal.2002581

    Figure Lengend Snippet: EGFR interaction with TLR3 is independent of TLR3 tyrosine residues and TRIF

    Article Snippet: Antibodies against Src, phospho-Tyr416 -Src and LRRK1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively; anti-human TLR3 (ab13915) and anti-mouse TLR3 (ab53424) were from Abcam (Cambridge, MA, USA); anti-human TRIF (4596), anti-EGFR, anti-ErbB2, and anti-phospho-Ser396 IRF-3 were from Cell Signaling (Danvers, MA, USA); anti-mouse TRIF (LS-C749/22434) were from LifeSpanBioSciences (Seattle, WA, USA); antibodies against IRF-3, His, and GST were from Santa Cruz Biotechnology; anti-phosphotyrosine (4G10 platinum) was from Millipore (Billerica, MA, USA); antibodies against P54, P56, P60, and DRBP76 were raised in our laboratory.

    Techniques:

    Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR

    Journal: Science signaling

    Article Title: EGFR is Essential for TLR3 Signaling

    doi: 10.1126/scisignal.2002581

    Figure Lengend Snippet: Src interaction with TLR3 is independent of TRIF and TRAF3, but dependent on EGFR

    Article Snippet: Antibodies against Src, phospho-Tyr416 -Src and LRRK1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA, USA), respectively; anti-human TLR3 (ab13915) and anti-mouse TLR3 (ab53424) were from Abcam (Cambridge, MA, USA); anti-human TRIF (4596), anti-EGFR, anti-ErbB2, and anti-phospho-Ser396 IRF-3 were from Cell Signaling (Danvers, MA, USA); anti-mouse TRIF (LS-C749/22434) were from LifeSpanBioSciences (Seattle, WA, USA); antibodies against IRF-3, His, and GST were from Santa Cruz Biotechnology; anti-phosphotyrosine (4G10 platinum) was from Millipore (Billerica, MA, USA); antibodies against P54, P56, P60, and DRBP76 were raised in our laboratory.

    Techniques:

    Levels of TLR4 and adaptor proteins during acquisition of resistance to docetaxel. Immunoblots were performed from extracts of MCF-7 and A2780 drug-naive and drug-resistant cell lines (A and B) in order to confirm the presence or absence of TLR4 and adaptor proteins MyD88 and TRIF. Changes in protein levels of drug-naive and drug-resistant cell lines were assessed by densitometry (C, D, E, and F). Statistical analysis consisted of two-tailed T-tests; *p

    Journal: PLoS ONE

    Article Title: Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance

    doi: 10.1371/journal.pone.0183662

    Figure Lengend Snippet: Levels of TLR4 and adaptor proteins during acquisition of resistance to docetaxel. Immunoblots were performed from extracts of MCF-7 and A2780 drug-naive and drug-resistant cell lines (A and B) in order to confirm the presence or absence of TLR4 and adaptor proteins MyD88 and TRIF. Changes in protein levels of drug-naive and drug-resistant cell lines were assessed by densitometry (C, D, E, and F). Statistical analysis consisted of two-tailed T-tests; *p

    Article Snippet: The membrane was blocked in 5% skim milk in TBST [0.24% Trizma® Base (Sigma), 0.8% NaCl (Fisher), 0.1% Tween20® (Sigma) at pH 7.6] for 1 hour before incubation overnight at 4°C with a human TLR4 antibody (1:250, Santa Cruz, Dallas, TX), human MyD88 antibody (1:1000, Cell Signaling, Danvers, MA), human TRIF antibody (1:700, Cell Signaling, Danvers, MA) or a GAPDH antibody (1:10,000, Santa Cruz).

    Techniques: Western Blot, Two Tailed Test

    Kinase activity of TBK1 is required for the recruitment of IRF-3 to TRIF in response to TLR3 stimulation. (A) WT and TBK1 KO 293T cells were transfected with expression constructs for HA-tagged TLR3 and Flag-tagged IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting for Flag and endogenous TRIF as indicated. Input, total lysates. (B) TBK1 KO 293T cells were stably reconstituted with empty vector or expression vector for mCherry-tagged WT TBK1 or TBK1 K38A and were transfected with expression constructs for Flag-tagged TLR3 and HA-IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting as indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling

    doi: 10.1128/MCB.00509-19

    Figure Lengend Snippet: Kinase activity of TBK1 is required for the recruitment of IRF-3 to TRIF in response to TLR3 stimulation. (A) WT and TBK1 KO 293T cells were transfected with expression constructs for HA-tagged TLR3 and Flag-tagged IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting for Flag and endogenous TRIF as indicated. Input, total lysates. (B) TBK1 KO 293T cells were stably reconstituted with empty vector or expression vector for mCherry-tagged WT TBK1 or TBK1 K38A and were transfected with expression constructs for Flag-tagged TLR3 and HA-IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting as indicated.

    Article Snippet: Rabbit monoclonal anti-IKKβ (2370), rabbit monoclonal anti-phospho TBK1 (Ser172) (5483), and rabbit monoclonal anti-TRIF (4596) were purchased from Cell Signaling Technology.

    Techniques: Activity Assay, Transfection, Expressing, Construct, Immunoprecipitation, Stable Transfection, Plasmid Preparation

    IKKβ plays an essential role in IRF-3 activation via TLR3/4 signaling but not RIG-I signaling. (A) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and HA-tagged IRF-3 S386A. Twenty-four hours later, cells were pretreated with 20 μM TPCA-1 or 10 μM BX795 for 1 h. The cells were stimulated with 50 μg/ml of poly(I·C) for 2 h with or without inhibitors as indicated. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA, followed by immunoblotting with anti-HA or anti-TRIF. (B) THP-1 cells stably expressing HA-tagged IRF-3 S386A were cultured in medium containing 10 ng/ml of PMA for 72 h. Cells were stimulated with 1 μg/ml of LPS for 2 h. Control solvent (DMSO), TPCA-1 (20 μM), or BX795 (10 μM) was added 1 h before stimulation and left until the end of the experiment. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA, followed by immunoblotting as indicated. *, nonspecific band. Input, total lysates. (C) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were pretreated with TPCA-1 at the indicated dose for 1 h. Cells were stimulated with 50 μg/ml of poly(I·C) for 12 h in the presence of TPCA-1. (D) 293T cells were transfected with reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were pretreated with TPCA-1 at the indicated dose for 1 h. Cells were infected with Sendai virus (SeV) for 24 h in the presence of TPCA. Cell lysates were prepared and subjected to dual-luciferase assay. (E and F) WT 293T cells and IKKα, IKKβ, IKKα/β, NEMO, TBK1, IRF-3, and TRIF KO 293T cells were transfected with expression constructs for Flag-tagged TLR3 (E) and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 12 h (E) or infected with SeV for 24 h (F). Cell lysates were prepared and subjected to dual-luciferase assay. (G) WT 293T cells and IKKα, IKKβ, and IKKα/β KO 293T cells were transfected with expression constructs for HA-tagged TLR3 and Flag-tagged IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting. (H) WT and IKKβ KO 293T cells were transfected with expression constructs for Flag-tagged TLR3 and HA-IRF-3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Total cell lysates were subjected to native PAGE and SDS-PAGE, followed by immunoblotting. *, nonspecific band. (I) WT 293T cells and IKKα, IKKβ, or IKKα/β KO 293T cells were infected with SeV for 8 h. Total cell lysates were subjected to native PAGE and SDS-PAGE, followed by immunoblotting. (J) WT and IKKβ KO 293T cells were stably reconstituted for IKKβ or IKKβ K44A. The cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 12 h. Cells were lysed and subjected to dual-luciferase assay. Luciferase activity data are representative results from at least three independent experiments; means and SD from triplicate experiments are shown. ** * , P

    Journal: Molecular and Cellular Biology

    Article Title: Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling

    doi: 10.1128/MCB.00509-19

    Figure Lengend Snippet: IKKβ plays an essential role in IRF-3 activation via TLR3/4 signaling but not RIG-I signaling. (A) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and HA-tagged IRF-3 S386A. Twenty-four hours later, cells were pretreated with 20 μM TPCA-1 or 10 μM BX795 for 1 h. The cells were stimulated with 50 μg/ml of poly(I·C) for 2 h with or without inhibitors as indicated. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA, followed by immunoblotting with anti-HA or anti-TRIF. (B) THP-1 cells stably expressing HA-tagged IRF-3 S386A were cultured in medium containing 10 ng/ml of PMA for 72 h. Cells were stimulated with 1 μg/ml of LPS for 2 h. Control solvent (DMSO), TPCA-1 (20 μM), or BX795 (10 μM) was added 1 h before stimulation and left until the end of the experiment. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA, followed by immunoblotting as indicated. *, nonspecific band. Input, total lysates. (C) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were pretreated with TPCA-1 at the indicated dose for 1 h. Cells were stimulated with 50 μg/ml of poly(I·C) for 12 h in the presence of TPCA-1. (D) 293T cells were transfected with reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were pretreated with TPCA-1 at the indicated dose for 1 h. Cells were infected with Sendai virus (SeV) for 24 h in the presence of TPCA. Cell lysates were prepared and subjected to dual-luciferase assay. (E and F) WT 293T cells and IKKα, IKKβ, IKKα/β, NEMO, TBK1, IRF-3, and TRIF KO 293T cells were transfected with expression constructs for Flag-tagged TLR3 (E) and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 12 h (E) or infected with SeV for 24 h (F). Cell lysates were prepared and subjected to dual-luciferase assay. (G) WT 293T cells and IKKα, IKKβ, and IKKα/β KO 293T cells were transfected with expression constructs for HA-tagged TLR3 and Flag-tagged IRF-3 S386A. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Cell lysates were subjected to immunoprecipitation with anti-HA, followed by immunoblotting. (H) WT and IKKβ KO 293T cells were transfected with expression constructs for Flag-tagged TLR3 and HA-IRF-3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 2 h. Total cell lysates were subjected to native PAGE and SDS-PAGE, followed by immunoblotting. *, nonspecific band. (I) WT 293T cells and IKKα, IKKβ, or IKKα/β KO 293T cells were infected with SeV for 8 h. Total cell lysates were subjected to native PAGE and SDS-PAGE, followed by immunoblotting. (J) WT and IKKβ KO 293T cells were stably reconstituted for IKKβ or IKKβ K44A. The cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55C1B luc and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for 12 h. Cells were lysed and subjected to dual-luciferase assay. Luciferase activity data are representative results from at least three independent experiments; means and SD from triplicate experiments are shown. ** * , P

    Article Snippet: Rabbit monoclonal anti-IKKβ (2370), rabbit monoclonal anti-phospho TBK1 (Ser172) (5483), and rabbit monoclonal anti-TRIF (4596) were purchased from Cell Signaling Technology.

    Techniques: Activation Assay, Transfection, Expressing, Construct, Immunoprecipitation, Stable Transfection, Cell Culture, Infection, Luciferase, Clear Native PAGE, SDS Page, Activity Assay

    IKKβ targets TBK1 to facilitate IRF-3 recruitment to TRIF and signaling. (A) 293T cells were transfected with expression constructs for Flag-tagged TRIF-N540, together with mCherry-tagged TBK1 WT/K38A or IKKβ WT/K44A. Twenty-four hours later, cell lysates were prepared and subjected to Phos-tag SDS-PAGE and normal SDS-PAGE, followed by immunoblotting as indicated. (B) IKKβ KO 293T cells stably reconstituted with empty vector, IKKβ, or IKKβ K44A were transfected with the expression construct for Flag-tagged TLR3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-TBK1, followed by immunoblotting. (C) TBK1 KO 293T cells stably reconstituted with mCherry-tagged TBK1 K38A were transfected with expression constructs for IKKβ or IKKβ K44A as indicated. Twenty-four hours later, cell lysates were prepared and subjected to immunoblotting. (D) TBK1 KO 293T cells stably reconstituted with mCherry-tagged TBK1 K38A were transfected with constructs for Flag-tagged TLR3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Where indicated, TPCA-1 (20 μM) was added 1 h before and left during stimulation. Total cell lysates were subjected to immunoblotting. (E) WT, IKKβ KO, and TBK1 KO 293T cells were infected with Sendai virus (SeV) for the indicated times. Total cell lysates were subjected to immunoblotting. (F and G) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55A2 luc [F] or p-55C1B luc [G] and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) together with 30 ng/ml of recombinant TNF-α for 12 h. Where indicated, TPCA-1 (20 μM) was added 1 h before and left during stimulation. Cells were lysed and subjected to dual-luciferase assay. Luciferase activity data are representative results from at least three independent experiments; means and SD from triplicate experiments are shown. * , P

    Journal: Molecular and Cellular Biology

    Article Title: Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling

    doi: 10.1128/MCB.00509-19

    Figure Lengend Snippet: IKKβ targets TBK1 to facilitate IRF-3 recruitment to TRIF and signaling. (A) 293T cells were transfected with expression constructs for Flag-tagged TRIF-N540, together with mCherry-tagged TBK1 WT/K38A or IKKβ WT/K44A. Twenty-four hours later, cell lysates were prepared and subjected to Phos-tag SDS-PAGE and normal SDS-PAGE, followed by immunoblotting as indicated. (B) IKKβ KO 293T cells stably reconstituted with empty vector, IKKβ, or IKKβ K44A were transfected with the expression construct for Flag-tagged TLR3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-TBK1, followed by immunoblotting. (C) TBK1 KO 293T cells stably reconstituted with mCherry-tagged TBK1 K38A were transfected with expression constructs for IKKβ or IKKβ K44A as indicated. Twenty-four hours later, cell lysates were prepared and subjected to immunoblotting. (D) TBK1 KO 293T cells stably reconstituted with mCherry-tagged TBK1 K38A were transfected with constructs for Flag-tagged TLR3. Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) for the indicated times. Where indicated, TPCA-1 (20 μM) was added 1 h before and left during stimulation. Total cell lysates were subjected to immunoblotting. (E) WT, IKKβ KO, and TBK1 KO 293T cells were infected with Sendai virus (SeV) for the indicated times. Total cell lysates were subjected to immunoblotting. (F and G) 293T cells were transfected with expression constructs for Flag-tagged TLR3 and reporter genes (p-55A2 luc [F] or p-55C1B luc [G] and pRL-TK Renilla luc). Twenty-four hours later, cells were stimulated with 50 μg/ml of poly(I·C) together with 30 ng/ml of recombinant TNF-α for 12 h. Where indicated, TPCA-1 (20 μM) was added 1 h before and left during stimulation. Cells were lysed and subjected to dual-luciferase assay. Luciferase activity data are representative results from at least three independent experiments; means and SD from triplicate experiments are shown. * , P

    Article Snippet: Rabbit monoclonal anti-IKKβ (2370), rabbit monoclonal anti-phospho TBK1 (Ser172) (5483), and rabbit monoclonal anti-TRIF (4596) were purchased from Cell Signaling Technology.

    Techniques: Transfection, Expressing, Construct, SDS Page, Stable Transfection, Plasmid Preparation, Immunoprecipitation, Infection, Recombinant, Luciferase, Activity Assay

    siRNA against TRIF, MDA5, but not RIG-I reduced RV1B-induced IRF3 dimerization

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell responses

    doi: 10.4049/jimmunol.0901386

    Figure Lengend Snippet: siRNA against TRIF, MDA5, but not RIG-I reduced RV1B-induced IRF3 dimerization

    Article Snippet: Membranes were blocked in 5% milk for 1 h at room temperature and probed with either mouse anti-RIG-I (Alexis Biochemicals, Plymouth Meeting, PA), goat anti-MDA5 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-IRF3 (IBL America, Minneapolis, MN), rabbit anti-TRIF (Cell Signaling, Danvers, MA) or rabbit anti-IRF7 (Abcam, Cambridge, MA).

    Techniques:

    MyD88 is required for inflammasome sensing of HIV and HCV. Knockdowns of the TLR adaptors MyD88 ( A , B ) and TICAM-1 (TRIF, E , F ) were generated and confirmed by previously described RNA interference techniques. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which MyD88 was knocked down were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( C , G ) and IL-18 secretion measured at 24 h ( D , H ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in MyD88 ( C , D ) or TICAM-1 ( G , H ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.001 compared to the scramble siRNA transfected cells.

    Journal: PLoS Pathogens

    Article Title: HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

    doi: 10.1371/journal.ppat.1004082

    Figure Lengend Snippet: MyD88 is required for inflammasome sensing of HIV and HCV. Knockdowns of the TLR adaptors MyD88 ( A , B ) and TICAM-1 (TRIF, E , F ) were generated and confirmed by previously described RNA interference techniques. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which MyD88 was knocked down were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( C , G ) and IL-18 secretion measured at 24 h ( D , H ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in MyD88 ( C , D ) or TICAM-1 ( G , H ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.001 compared to the scramble siRNA transfected cells.

    Article Snippet: Antibodies Antibodies anti-Human CD81 mouse mAb (clone 1.3.3.22, Santa Cruz Biotechnology, Santa Cruz CA); Anti-human CD4 mouse mAb (clone SK3) and an isotype control (clone MOPC-21, Biolegend, San Diego CA); Anti-human TLR3 rabbit mAb (clone D10F10, Cell Signaling Technology, Danvers MA); Anti-human TLR7 rabbit pAb (#2633S, Cell signaling Technology); Anti-human TLR8 rabbit mAb (clone D3Z6J, Cell Signaling Technology); Anti-human TLR9 rabbit mAb (clone D2C9, Cell Signaling Technology); Anti-human MyD88 rabbit mAb (clone D80F5, Cell Signaling Technology); Anti-human TRIF/TICAM-1 (clone MAB6216, R & D Systems, Minneapolis MN) mouse mAb; Anti-human NALP3 rabbit pAb (Imgenex, San Diego CA); Anti-human RIG-I (D14G6) rabbit mAb (clone D14G6, Cell Signaling Technology); Anti-human AIM2 rabbit pAb (Abcam, Cambridge, MA) and Anti-human Actin (A2066, Sigma-Aldrich) were purchased from respective vendors.

    Techniques: Generated, Transfection, Cell Culture

    Effects of asunaprevir on replication of HCV in JFH-1- infected . Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. JFH-1-infected Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. HCV core protein, MAVS and TRIF were determined by immunoblotting analysis. The HCV core protein levels relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in the figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P

    Journal: Frontiers in Microbiology

    Article Title: Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus

    doi: 10.3389/fmicb.2017.00668

    Figure Lengend Snippet: Effects of asunaprevir on replication of HCV in JFH-1- infected . Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. JFH-1-infected Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. HCV core protein, MAVS and TRIF were determined by immunoblotting analysis. The HCV core protein levels relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in the figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P

    Article Snippet: The primary antibodies used were anti-STAT1, anti-phospho-STAT1, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO).

    Techniques: Infection, Transfection, Western Blot, Software

    Effects of Asunaprevir on TLR3/RIG-I signaling pathway in Huh. 7.5.1 cells with MAVS and TRIF knockdown . Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. The key signaling proteins such as MAVS, TRIF, IRF3, and phosphorylated IRF-3 were determined by immunoblotting analysis (left panel). Immunoblots shown in the figure are representative of three independent experiments. The protein levels phosphorylated IRF-3 over total IRF3 was analyzed with ImageJ software (right panel). Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P

    Journal: Frontiers in Microbiology

    Article Title: Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus

    doi: 10.3389/fmicb.2017.00668

    Figure Lengend Snippet: Effects of Asunaprevir on TLR3/RIG-I signaling pathway in Huh. 7.5.1 cells with MAVS and TRIF knockdown . Huh 7.5.1 cells were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir for 24 h. The key signaling proteins such as MAVS, TRIF, IRF3, and phosphorylated IRF-3 were determined by immunoblotting analysis (left panel). Immunoblots shown in the figure are representative of three independent experiments. The protein levels phosphorylated IRF-3 over total IRF3 was analyzed with ImageJ software (right panel). Data are mean ± SD from 3 independent tests. Statistical significance was tested by Student's t -test, * P

    Article Snippet: The primary antibodies used were anti-STAT1, anti-phospho-STAT1, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO).

    Techniques: Transfection, Western Blot, Software

    Effects of asunaprevir on replication of DENV in Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. (A) Huh 7.5.1 cells infected with DENV were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir. Immunoblotting analysis for NS3 protein of DENV, MAVS, and TRIF were determined by immunoblotting Analysis (upper panels). The DENV NS3 protein levels relative to the β-actin were shown at the bottom of each sample. Densitometry was performed with ImageJ software (lower panel). Data are mean ± SD from 3 independent tests. Student's t -test was used as statistical test, *** P

    Journal: Frontiers in Microbiology

    Article Title: Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus

    doi: 10.3389/fmicb.2017.00668

    Figure Lengend Snippet: Effects of asunaprevir on replication of DENV in Huh 7.5.1 cells after knockdown of MAVS and TRIF by siRNA. (A) Huh 7.5.1 cells infected with DENV were transfected by siRNA of MAVS and TRIF for 48 h and then treated with asunaprevir. Immunoblotting analysis for NS3 protein of DENV, MAVS, and TRIF were determined by immunoblotting Analysis (upper panels). The DENV NS3 protein levels relative to the β-actin were shown at the bottom of each sample. Densitometry was performed with ImageJ software (lower panel). Data are mean ± SD from 3 independent tests. Student's t -test was used as statistical test, *** P

    Article Snippet: The primary antibodies used were anti-STAT1, anti-phospho-STAT1, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO).

    Techniques: Infection, Transfection, Software

    Asunaprevir activates ISRE activity and type I IFN and TLR3/RIG-I antiviral signaling pathway. (A) ISRE luciferase reporter assay, plasmids of pISRE-luc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1 or 10 nM of asunaprevir for 3, 6, 24, and 48 h. The 0 nM indicated DMSO vehicle control. The firefly and Renilla luciferase activities were then measured by dual-luciferase assay. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (B) Huh 7.5.1 were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the interferon signaling pathway (upper panels). The protein levels of STAT-1, phosphorylated STAT-1, STAT-2, phosphorylated STAT-2, MxA, and ISG-15 relative to the β-actin were determined by densitometry with ImageJ software (lower, panels). (C) IFN-β luciferase reporter assay, plasmids pIFN-β/Fluc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1, 10, or 100 nM of asunaprevir for 24 h. Firefly and Renilla luciferase activities were then measured. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (D) Huh 7.5.1 cells were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the TLR3/RIG-I signaling pathway (upper panels). The protein levels of MAVS, TRIF, IRF-3, and phosphorylated IRF-3 relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in each figure are representative of three independent experiments. Densitometry was performed with ImageJ software (lower, panels). Values represent the average of three assays ± S.D . Statistical significance was tested by Student's t -test, * P

    Journal: Frontiers in Microbiology

    Article Title: Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus

    doi: 10.3389/fmicb.2017.00668

    Figure Lengend Snippet: Asunaprevir activates ISRE activity and type I IFN and TLR3/RIG-I antiviral signaling pathway. (A) ISRE luciferase reporter assay, plasmids of pISRE-luc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1 or 10 nM of asunaprevir for 3, 6, 24, and 48 h. The 0 nM indicated DMSO vehicle control. The firefly and Renilla luciferase activities were then measured by dual-luciferase assay. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (B) Huh 7.5.1 were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the interferon signaling pathway (upper panels). The protein levels of STAT-1, phosphorylated STAT-1, STAT-2, phosphorylated STAT-2, MxA, and ISG-15 relative to the β-actin were determined by densitometry with ImageJ software (lower, panels). (C) IFN-β luciferase reporter assay, plasmids pIFN-β/Fluc expressing firefly luciferase and pRL-TK expressing Renilla luciferase as an internal control were co-transfected to Huh 7.5.1 cells and then treated with 1, 10, or 100 nM of asunaprevir for 24 h. Firefly and Renilla luciferase activities were then measured. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (D) Huh 7.5.1 cells were treated with different doses of asunaprevir for 48 h and the cell lysates were analyzed by immunoblotting with the indicated antibodies involved in the TLR3/RIG-I signaling pathway (upper panels). The protein levels of MAVS, TRIF, IRF-3, and phosphorylated IRF-3 relative to the β-actin were shown at the bottom of each sample. Immunoblots shown in each figure are representative of three independent experiments. Densitometry was performed with ImageJ software (lower, panels). Values represent the average of three assays ± S.D . Statistical significance was tested by Student's t -test, * P

    Article Snippet: The primary antibodies used were anti-STAT1, anti-phospho-STAT1, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO).

    Techniques: Activity Assay, Luciferase, Reporter Assay, Expressing, Transfection, Software, Western Blot