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  • 95
    Name:
    TRIF Antibody
    Description:
    Members of the Toll like receptor TLR family named for the closely related Toll receptor in Drosophila play a pivotal role in innate immune responses 1 4 TLRs recognize conserved motifs found in various pathogens and mediate defense responses 5 7 Triggering of the TLR pathway leads to the activation of NF κB and subsequent regulation of immune and inflammatory genes 4 The TLRs and members of the IL 1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll Interleukin 1 receptor TIR domain 1 Upon activation TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains including myeloid differentiation factor 88 MyD88 MyD88 adaptor like TIR associated protein MAL TIRAP Toll receptor associated activator of interferon TRIF and Toll receptor associated molecule TRAM 8 10 This association leads to the recruitment and activation of IRAK1 and IRAK4 which form a complex with TRAF6 to activate TAK1 and IKK 8 11 14 Activation of IKK leads to the degradation of IκB which normally maintains NF κB in an inactive state by sequestering it in the cytoplasm TRIF also termed TICAM 1 is a TIR domain adaptor protein described to activate NF κB IRF3 and trigger IFN β production 4 5 Studies using dominant negative forms of TRIF and siRNA targeting TRIF show that TRIF functions downstream of both TLR3 and TLR4 in response to dsRNA and LPS respectively 4 6 TRIF recruits TRAF6 TAK1 TAB2 to the receptor complex which leads to NF κB activation 7 In addition TRIF can trigger signaling of that lead to the induction of apoptosis 8
    Catalog Number:
    4596
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser219 of human TRIF/TICAM-1. Antibodies were purified by peptide affinity chromatography.
    Reactivity:
    Human
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc trif
    Silencing <t>RIG-I</t> subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), <t>Trif</t> or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.
    Members of the Toll like receptor TLR family named for the closely related Toll receptor in Drosophila play a pivotal role in innate immune responses 1 4 TLRs recognize conserved motifs found in various pathogens and mediate defense responses 5 7 Triggering of the TLR pathway leads to the activation of NF κB and subsequent regulation of immune and inflammatory genes 4 The TLRs and members of the IL 1 receptor family share a conserved stretch of approximately 200 amino acids known as the Toll Interleukin 1 receptor TIR domain 1 Upon activation TLRs associate with a number of cytoplasmic adaptor proteins containing TIR domains including myeloid differentiation factor 88 MyD88 MyD88 adaptor like TIR associated protein MAL TIRAP Toll receptor associated activator of interferon TRIF and Toll receptor associated molecule TRAM 8 10 This association leads to the recruitment and activation of IRAK1 and IRAK4 which form a complex with TRAF6 to activate TAK1 and IKK 8 11 14 Activation of IKK leads to the degradation of IκB which normally maintains NF κB in an inactive state by sequestering it in the cytoplasm TRIF also termed TICAM 1 is a TIR domain adaptor protein described to activate NF κB IRF3 and trigger IFN β production 4 5 Studies using dominant negative forms of TRIF and siRNA targeting TRIF show that TRIF functions downstream of both TLR3 and TLR4 in response to dsRNA and LPS respectively 4 6 TRIF recruits TRAF6 TAK1 TAB2 to the receptor complex which leads to NF κB activation 7 In addition TRIF can trigger signaling of that lead to the induction of apoptosis 8
    https://www.bioz.com/result/trif/product/Cell Signaling Technology Inc
    Average 95 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    trif - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection"

    Article Title: Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection

    Journal: BMC Immunology

    doi: 10.1186/s12865-014-0041-4

    Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.
    Figure Legend Snippet: Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.

    Techniques Used: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Concentration Assay, Infection

    2) Product Images from "TLR2 signaling pathway combats Streptococcus uberis infection by inducing production of mitochondrial reactive oxygen species"

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

    Journal: bioRxiv

    doi: 10.1101/809186

    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
    Figure Legend 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

    Techniques Used: Infection, Immunohistochemistry, Expressing

    3) Product Images from "The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling"

    Article Title: The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001311

    3C pro Cleaves MAVS and TRIF. ( A ) Immunoblot analysis for Flag-MAVS (top) and GFP (bottom) in HEK293 cells co-transfected with EGFP-2B, 2C, 3A, or 3C pro and Flag-MAVS. In order to better visualize cleavage fragments, EGFP-3C pro expressing cells were transfected with 2 µg Flag-MAVS (in comparison to 1 µg with other constructs). ( B ) HEK293 cells transfected with Flag-MAVS and control (EGFP-C2), EGFP-3Cpro wild-type or C147A were lysed and subjected to immunoblotting for MAVS and GFP. ( C ) 3C pro cleaves MAVS in vitro . Recombinant wild-type or C147A mutant SUMO-3C pro (10 µg) was incubated with column purified Flag-MAVS (0.1 µg) for 8 hrs at 37°C, fractionated by SDS-PAGE, and immunoblotted with anti-Flag monoclonal antibody (top) or commassie stained (bottom). ( D ) U2OS cells were transfected with EGFP-3C pro wild-type (WT) or the C147A mutant and Flag-MAVS and immunofluorescence microscopy performed for MAVS (in red). ( E ) Immunoblot analysis of HEK293 cells transfected with CFP-TRIF and either vector control, EGFP-2A pro , 3A EGFP-3C pro and lysates immunoblotted for TRIF (top) or GFP (bottom). Grey arrows denote 3C pro -induced cleavage products. ( F ) Lysates of HEK293 cells transfected with CFP-TRIF and vector, wild-type (WT), or C147A mutant EGFP-3C pro were immunoblotted with anti- TRIF antibody (top) or anti-GFP (bottom) antibodies. Grey arrow denotes 3C pro -induced cleavage product. ( G ) 3C pro cleaves TRIF in vitro . Recombinant wild-type or C147A mutant SUMO-3C pro (10 µg) was incubated with column purified Flag-TRIF (0.1 µg) for 12 hrs at 37°C, fractionated by SDS-PAGE, and immunoblotted with anti-Flag monoclonal antibody (top) or commassie stained (bottom).
    Figure Legend Snippet: 3C pro Cleaves MAVS and TRIF. ( A ) Immunoblot analysis for Flag-MAVS (top) and GFP (bottom) in HEK293 cells co-transfected with EGFP-2B, 2C, 3A, or 3C pro and Flag-MAVS. In order to better visualize cleavage fragments, EGFP-3C pro expressing cells were transfected with 2 µg Flag-MAVS (in comparison to 1 µg with other constructs). ( B ) HEK293 cells transfected with Flag-MAVS and control (EGFP-C2), EGFP-3Cpro wild-type or C147A were lysed and subjected to immunoblotting for MAVS and GFP. ( C ) 3C pro cleaves MAVS in vitro . Recombinant wild-type or C147A mutant SUMO-3C pro (10 µg) was incubated with column purified Flag-MAVS (0.1 µg) for 8 hrs at 37°C, fractionated by SDS-PAGE, and immunoblotted with anti-Flag monoclonal antibody (top) or commassie stained (bottom). ( D ) U2OS cells were transfected with EGFP-3C pro wild-type (WT) or the C147A mutant and Flag-MAVS and immunofluorescence microscopy performed for MAVS (in red). ( E ) Immunoblot analysis of HEK293 cells transfected with CFP-TRIF and either vector control, EGFP-2A pro , 3A EGFP-3C pro and lysates immunoblotted for TRIF (top) or GFP (bottom). Grey arrows denote 3C pro -induced cleavage products. ( F ) Lysates of HEK293 cells transfected with CFP-TRIF and vector, wild-type (WT), or C147A mutant EGFP-3C pro were immunoblotted with anti- TRIF antibody (top) or anti-GFP (bottom) antibodies. Grey arrow denotes 3C pro -induced cleavage product. ( G ) 3C pro cleaves TRIF in vitro . Recombinant wild-type or C147A mutant SUMO-3C pro (10 µg) was incubated with column purified Flag-TRIF (0.1 µg) for 12 hrs at 37°C, fractionated by SDS-PAGE, and immunoblotted with anti-Flag monoclonal antibody (top) or commassie stained (bottom).

    Techniques Used: Transfection, Expressing, Construct, In Vitro, Recombinant, Mutagenesis, Incubation, Purification, SDS Page, Staining, Immunofluorescence, Microscopy, Plasmid Preparation

    CVB3 infection induces MAVS and TRIF cleavage. ( A ) Western blot analysis for MAVS in HEK293 cells infected with CVB3 for 12 hrs in the absence (NoI) or presence of Z-VAD-FMK (zVAD) or MG132. ( B ) Time course of MAVS and caspase-3 cleavage in HEK293 cells infected with CVB3 for the indicated times. ( C ) HEK293 cells were infected with CVB3 for 8 hrs and fixed and stained for MAVS (green), mitochondria (red), and VP1 (blue). Asterisks denote infected cells expressing less MAVS than uninfected controls. ( D , E ) U2OS cells transfected with Flag-MAVS or EGFP-MAVS were infected with CVB3 (1 PFU/cell) for 7 hrs (D) or 12 hrs (E) and then lysed and immunoblotted with anti-Flag monoclonal antibody (D) or fixed and stained for mitochondria (red) and VP1 (blue) (E). In order to better visualize cleavage fragments in (D), CVB-infected cultures were transfected with 2 µg Flag-MAVS (in comparison to 1 µg in uninfected controls). ( F ) Western blot analysis for TRIF in HeLa cells infected with CVB3 for 8 hrs in the absence (NoI) or presence of z-VAD-FMK (zVAD) or MG132. ( G ) Time course of TRIF cleavage in HeLa cells infected with CVB3 (1 PFU/cell) for the indicated times. ( H ) Immunoblot analysis for overexpressed CFP-TRIF in HEK293 cells infected with CVB3 (1 PFU/cell) for 8 hrs. In (A), (D), and (H), grey arrows denote CVB3-induced cleavage fragments.
    Figure Legend Snippet: CVB3 infection induces MAVS and TRIF cleavage. ( A ) Western blot analysis for MAVS in HEK293 cells infected with CVB3 for 12 hrs in the absence (NoI) or presence of Z-VAD-FMK (zVAD) or MG132. ( B ) Time course of MAVS and caspase-3 cleavage in HEK293 cells infected with CVB3 for the indicated times. ( C ) HEK293 cells were infected with CVB3 for 8 hrs and fixed and stained for MAVS (green), mitochondria (red), and VP1 (blue). Asterisks denote infected cells expressing less MAVS than uninfected controls. ( D , E ) U2OS cells transfected with Flag-MAVS or EGFP-MAVS were infected with CVB3 (1 PFU/cell) for 7 hrs (D) or 12 hrs (E) and then lysed and immunoblotted with anti-Flag monoclonal antibody (D) or fixed and stained for mitochondria (red) and VP1 (blue) (E). In order to better visualize cleavage fragments in (D), CVB-infected cultures were transfected with 2 µg Flag-MAVS (in comparison to 1 µg in uninfected controls). ( F ) Western blot analysis for TRIF in HeLa cells infected with CVB3 for 8 hrs in the absence (NoI) or presence of z-VAD-FMK (zVAD) or MG132. ( G ) Time course of TRIF cleavage in HeLa cells infected with CVB3 (1 PFU/cell) for the indicated times. ( H ) Immunoblot analysis for overexpressed CFP-TRIF in HEK293 cells infected with CVB3 (1 PFU/cell) for 8 hrs. In (A), (D), and (H), grey arrows denote CVB3-induced cleavage fragments.

    Techniques Used: Infection, Western Blot, Staining, Expressing, Transfection

    3C pro abrogates MAVS and TRIF Type I IFN and apoptotic signaling. ( A , B ) Luciferase assay (expressed as fold IFNβ induction versus vector controls) from HEK293 cells transfected with vector, EGFP-3C pro wild-type or C147A mutants, the CARDs of MAVS, MDA5, or RIG-I, and a IFNβ promoted luciferase construct. Data are shown as mean ± standard deviation. Asterisks indicate p-values ≤0.05 ( C ) Representative images of either untransfected (No Tx) U2OS cells or cells transfected with Flag-MAVS (top row) or TRIF (bottom row) and either vector alone (+Vector) or wild-type (WT) EGFP-3C pro . Cells were stained with Alexa Fluor 594-conjugated Annexin V 48 hrs post-transfection. Blue, DAPI-stained nuclei. ( D ) Quantification of the extent of apoptosis (shown as the percent of Annexin V positive cells/DAPI) in cells from (C). Asterisks indicate p-values ≤0.05.
    Figure Legend Snippet: 3C pro abrogates MAVS and TRIF Type I IFN and apoptotic signaling. ( A , B ) Luciferase assay (expressed as fold IFNβ induction versus vector controls) from HEK293 cells transfected with vector, EGFP-3C pro wild-type or C147A mutants, the CARDs of MAVS, MDA5, or RIG-I, and a IFNβ promoted luciferase construct. Data are shown as mean ± standard deviation. Asterisks indicate p-values ≤0.05 ( C ) Representative images of either untransfected (No Tx) U2OS cells or cells transfected with Flag-MAVS (top row) or TRIF (bottom row) and either vector alone (+Vector) or wild-type (WT) EGFP-3C pro . Cells were stained with Alexa Fluor 594-conjugated Annexin V 48 hrs post-transfection. Blue, DAPI-stained nuclei. ( D ) Quantification of the extent of apoptosis (shown as the percent of Annexin V positive cells/DAPI) in cells from (C). Asterisks indicate p-values ≤0.05.

    Techniques Used: Luciferase, Plasmid Preparation, Transfection, Construct, Standard Deviation, Staining

    4) Product Images from "Type I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors"

    Article Title: Type I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors

    Journal: Mucosal immunology

    doi: 10.1038/mi.2014.54

    MAVS is required for the gene expression of the majority of RSV inducible airway proteases. Wildtype, Trif ( Ticam1 ) and Mavs KO mice were infected with 1×10 6 pfu of RSV and animals were euthanized every second day as previously described. MMP and cathepsin gene expression in lung tissue was analyzed by qPCR on the day post infection with the maximal level of gene expression for each protease, as determined in Figure 2 . MMP-10 and -20 were examined one-day post infection. MMP-7, -8, -9 and cathepsin B were examined three days post infection. MMP-3, -16 and -17 were examined five days post infection. MMP-2, -12, -13, -14, -19, -25, -27, -28, cathepsin C, L1, S, W and Z were examined seven days post infection. Cathepsin E, G, H and K were examined nine days post infection. Results are represented as relative quantification (RQ) of the mean ± S.E.M when compared to mock treated animals on each corresponding day, with each measurement performed 3 times on 12 animals/group. p values shown, comparing treatments connected by a line.
    Figure Legend Snippet: MAVS is required for the gene expression of the majority of RSV inducible airway proteases. Wildtype, Trif ( Ticam1 ) and Mavs KO mice were infected with 1×10 6 pfu of RSV and animals were euthanized every second day as previously described. MMP and cathepsin gene expression in lung tissue was analyzed by qPCR on the day post infection with the maximal level of gene expression for each protease, as determined in Figure 2 . MMP-10 and -20 were examined one-day post infection. MMP-7, -8, -9 and cathepsin B were examined three days post infection. MMP-3, -16 and -17 were examined five days post infection. MMP-2, -12, -13, -14, -19, -25, -27, -28, cathepsin C, L1, S, W and Z were examined seven days post infection. Cathepsin E, G, H and K were examined nine days post infection. Results are represented as relative quantification (RQ) of the mean ± S.E.M when compared to mock treated animals on each corresponding day, with each measurement performed 3 times on 12 animals/group. p values shown, comparing treatments connected by a line.

    Techniques Used: Expressing, Mouse Assay, Infection, Real-time Polymerase Chain Reaction

    RSV induces the MAVS/MDA5/RIG-I/TLR3 pathways in mouse lungs. (A) Immunblots were performed to examine levels of MDA, RIG-I, MAVS, MyD88 and TRIF protein and IRF3 and TBK1 phosphorylation in lung tissue of RSV infected animals. Densitometry analysis was performed against actin or total protein (TBK1 and IRF3) levels for all samples from multiple immunoblots. (B) NFκB and AP-1 nuclear activity levels were determined in the lung tissue of mock and RSV treated mice. Graphs are represented as mean ± S.E.M, where each measurement performed 3 times on 12 animals/group. *Represents a p value less than 0.05 compared to mock treated mice on each corresponding day.
    Figure Legend Snippet: RSV induces the MAVS/MDA5/RIG-I/TLR3 pathways in mouse lungs. (A) Immunblots were performed to examine levels of MDA, RIG-I, MAVS, MyD88 and TRIF protein and IRF3 and TBK1 phosphorylation in lung tissue of RSV infected animals. Densitometry analysis was performed against actin or total protein (TBK1 and IRF3) levels for all samples from multiple immunoblots. (B) NFκB and AP-1 nuclear activity levels were determined in the lung tissue of mock and RSV treated mice. Graphs are represented as mean ± S.E.M, where each measurement performed 3 times on 12 animals/group. *Represents a p value less than 0.05 compared to mock treated mice on each corresponding day.

    Techniques Used: Multiple Displacement Amplification, Infection, Western Blot, Activity Assay, Mouse Assay

    5) Product Images from "miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose"

    Article Title: miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose

    Journal: Mediators of Inflammation

    doi: 10.1155/2016/3958453

    Changes of MyD88-independent signaling, TRIF and IRF3 levels, in REC. REC were cultured in normal glucose (5 mM, NG) or high glucose (25 mM, HG). Overexpression of miR-146a decreased the levels of TRIF (a) and IRF3 (b) in REC, compared to that of control HG group. A representative blot is shown. # p
    Figure Legend Snippet: Changes of MyD88-independent signaling, TRIF and IRF3 levels, in REC. REC were cultured in normal glucose (5 mM, NG) or high glucose (25 mM, HG). Overexpression of miR-146a decreased the levels of TRIF (a) and IRF3 (b) in REC, compared to that of control HG group. A representative blot is shown. # p

    Techniques Used: Cell Culture, Over Expression

    6) Product Images from "A Novel Agonist of the TRIF Pathway Induces a Cellular State Refractory to Replication of Zika, Chikungunya, and Dengue Viruses"

    Article Title: A Novel Agonist of the TRIF Pathway Induces a Cellular State Refractory to Replication of Zika, Chikungunya, and Dengue Viruses

    Journal: mBio

    doi: 10.1128/mBio.00452-17

    AV-C elicits an antiviral state in cells lacking the IPS-1/MAVS or STING pathways but not the TRIF or IFNAR pathways. (A) Average PFU per milliliter (± the standard deviation) for CHIKV, DENV, or ZIKV grown on wild-type (WT) THF-ISRE cells (blue) and THF-ISRE lacking IPS-1/MAVS (red), STING (gray), TRIF (black), or IFNAR (yellow) following 6 h pretreatment with 1% DMSO, 12.5 μM AV-C, or 1,000 U/ml IFN-β (the DMSO concentration was normalized to 1%). Infections were performed in triplicate, and virus titers were determined by serial dilution plaque assay at 48 h postinfection (CHIKV) or by FFU assay at 72 h postinfection (ZIKV and DENV) as described in the text. (B) Average units per milliliter of type I IFN, as determined in a luciferase bioassay of levels secreted from the indicated cells following 8-h treatment with 12.5 μM AV-C. (C) Average (in picograms per milliliter) ± the standard deviation of secreted IFN-β, determined by ELISA from human PBMCs following 8-h treatment in triplicate with the indicated concentrations of AV-C. An unpaired Student’s t test was used to determine statistical significance. *, P
    Figure Legend Snippet: AV-C elicits an antiviral state in cells lacking the IPS-1/MAVS or STING pathways but not the TRIF or IFNAR pathways. (A) Average PFU per milliliter (± the standard deviation) for CHIKV, DENV, or ZIKV grown on wild-type (WT) THF-ISRE cells (blue) and THF-ISRE lacking IPS-1/MAVS (red), STING (gray), TRIF (black), or IFNAR (yellow) following 6 h pretreatment with 1% DMSO, 12.5 μM AV-C, or 1,000 U/ml IFN-β (the DMSO concentration was normalized to 1%). Infections were performed in triplicate, and virus titers were determined by serial dilution plaque assay at 48 h postinfection (CHIKV) or by FFU assay at 72 h postinfection (ZIKV and DENV) as described in the text. (B) Average units per milliliter of type I IFN, as determined in a luciferase bioassay of levels secreted from the indicated cells following 8-h treatment with 12.5 μM AV-C. (C) Average (in picograms per milliliter) ± the standard deviation of secreted IFN-β, determined by ELISA from human PBMCs following 8-h treatment in triplicate with the indicated concentrations of AV-C. An unpaired Student’s t test was used to determine statistical significance. *, P

    Techniques Used: Standard Deviation, Concentration Assay, Serial Dilution, Plaque Assay, Luciferase, Enzyme-linked Immunosorbent Assay

    AV-C induces IRF3-mediated transcriptional activity in a manner independent of STING and IPS-1/MAVS but dependent on TRIF. (A) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking IPS1/MAVS (THF-ISRE-ΔIPS1) following 8-h treatment with SeV (160 HAU/ml) or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results with DMSO-treated cells of three replicates (± standard deviations [SD]). (B) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔIPS1 cells treated for 8 h with SeV (160 HAU/ml), or 50 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (C) Immunoblot showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔIPS1 cells left untreated and following 4-h exposure to 100 μM G10, SeV (160 HAU/ml), or 25 μM AV-C. (D) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking STING (THF-ISRE-ΔSTING) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (E) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔSTING cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes compared to results in DMSO-treated cells of two biological replicates, ± SD. (F) Immunoblot results showing the S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔSTING cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C. (G) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking TRIF (THF-ISRE-ΔTRIF) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (H) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔTRIF cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (I) Immunoblot results, showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔTRIF cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C.
    Figure Legend Snippet: AV-C induces IRF3-mediated transcriptional activity in a manner independent of STING and IPS-1/MAVS but dependent on TRIF. (A) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking IPS1/MAVS (THF-ISRE-ΔIPS1) following 8-h treatment with SeV (160 HAU/ml) or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results with DMSO-treated cells of three replicates (± standard deviations [SD]). (B) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔIPS1 cells treated for 8 h with SeV (160 HAU/ml), or 50 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (C) Immunoblot showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔIPS1 cells left untreated and following 4-h exposure to 100 μM G10, SeV (160 HAU/ml), or 25 μM AV-C. (D) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking STING (THF-ISRE-ΔSTING) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (E) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔSTING cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes compared to results in DMSO-treated cells of two biological replicates, ± SD. (F) Immunoblot results showing the S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔSTING cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C. (G) Reporter assay results, showing induction of ISRE-dependent LUC expression in THF-ISRE cells lacking TRIF (THF-ISRE-ΔTRIF) following 8-h treatment with 100 μM G10 or AV-C at the indicated concentrations. Values displayed are average fold changes compared to results in DMSO-treated cells of three replicates, ± SD. (H) Transcription of ISG15, IFIT1, viperin, and Mx2 in THF-ISRE-ΔTRIF cells treated for 8 h with 100 μM G10 or 25 μM AV-C. Values displayed are average fold changes versus results in DMSO-treated cells of two biological replicates, ± SD. (I) Immunoblot results, showing S386 phosphorylation status of IRF3 as well as total IRF3, TRIF, STING, IPS1/MAVS, and GAPDH in THF-ISRE-ΔTRIF cells left untreated and following 4-h exposure to 100 μM G10 or 25 μM AV-C.

    Techniques Used: Activity Assay, Reporter Assay, Expressing

    7) Product Images from "Hepatoprotective Effects of Kaempferol-3-O-α-l-Arabinopyranosyl-7-O-α-l-Rhamnopyranoside on d-Galactosamine and Lipopolysaccharide Caused Hepatic Failure in Mice"

    Article Title: Hepatoprotective Effects of Kaempferol-3-O-α-l-Arabinopyranosyl-7-O-α-l-Rhamnopyranoside on d-Galactosamine and Lipopolysaccharide Caused Hepatic Failure in Mice

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules22101755

    The expression of NF-κB and TLR4 signaling pathway-related proteins, such as NF-Κb ( A ); IκBα ( B ); p65 ( C ); TLR4 ( D ); TRAF6 ( E ); MyD88 ( F ); and TRIF ( G ); were detected using a Western blotting assay in the liver tissues of each group 6 h after D-GalN/LPS administration. The data represented show the mean ± SD. Data of IκBα, p65, and NF-κB are normalized to that of Lamin B, and that of TLR4, TRAF6, MyD88, and TRIF are normalized to β-actin. * p
    Figure Legend Snippet: The expression of NF-κB and TLR4 signaling pathway-related proteins, such as NF-Κb ( A ); IκBα ( B ); p65 ( C ); TLR4 ( D ); TRAF6 ( E ); MyD88 ( F ); and TRIF ( G ); were detected using a Western blotting assay in the liver tissues of each group 6 h after D-GalN/LPS administration. The data represented show the mean ± SD. Data of IκBα, p65, and NF-κB are normalized to that of Lamin B, and that of TLR4, TRAF6, MyD88, and TRIF are normalized to β-actin. * p

    Techniques Used: Expressing, Western Blot

    8) Product Images from "Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti‐inflammatory effects through blocking toll‐like receptor 4 dimerization) Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti‐inflammatory effects through blocking toll‐like receptor 4 dimerization"

    Article Title: Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti‐inflammatory effects through blocking toll‐like receptor 4 dimerization) Isoacteoside, a dihydroxyphenylethyl glycoside, exhibits anti‐inflammatory effects through blocking toll‐like receptor 4 dimerization

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13912

    Isoacteoside (ISO) blocked LPS‐induced TLR4 dimerization, which is involved in the MyD88 and TRIF pathway. (A) HEK293T cells were co‐transfected with TLR4‐HA and TLR‐Flag for 24 h. Cells were pretreated with isoacteoside (80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 section. Collected proteins were immunoprecipitated with anti‐HA magnetic beads. Immunocomplexes were determined by Western blotting with anti‐HA and anti‐Flag antibodies ( n = 5). (B, C) RAW264.7 cells were pretreated with isoacteoside (80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 ) for another 2 h. Collected proteins were immunoprecipitated with MyD88 or TRIF using magnetic beads. Immunocomplexes were determined by Western blotting with anti‐MyD88, anti‐TRIF and anti‐TLR4 antibodies ( n = 5). (D) RAW264.7 cells were pretreated with isoacteoside (20, 40 and 80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 ) for another 2 h. The p‐TAK1 and TAK1 expression was examined by Western blotting ( n = 5). * P
    Figure Legend Snippet: Isoacteoside (ISO) blocked LPS‐induced TLR4 dimerization, which is involved in the MyD88 and TRIF pathway. (A) HEK293T cells were co‐transfected with TLR4‐HA and TLR‐Flag for 24 h. Cells were pretreated with isoacteoside (80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 section. Collected proteins were immunoprecipitated with anti‐HA magnetic beads. Immunocomplexes were determined by Western blotting with anti‐HA and anti‐Flag antibodies ( n = 5). (B, C) RAW264.7 cells were pretreated with isoacteoside (80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 ) for another 2 h. Collected proteins were immunoprecipitated with MyD88 or TRIF using magnetic beads. Immunocomplexes were determined by Western blotting with anti‐MyD88, anti‐TRIF and anti‐TLR4 antibodies ( n = 5). (D) RAW264.7 cells were pretreated with isoacteoside (20, 40 and 80 μM) for 1 h before being stimulated with LPS (1 μg mL −1 ) for another 2 h. The p‐TAK1 and TAK1 expression was examined by Western blotting ( n = 5). * P

    Techniques Used: Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Expressing

    9) Product Images from "Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway "

    Article Title: Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway

    Journal: Journal of Virology

    doi: 10.1128/JVI.01250-09

    BV transduction activated the TLR3 signaling pathway. (A) Activation of TRIF, IRF-3, and NF-κB. (B) Nuclear translocation of IRF-3 and NF-κB. The nuclear and cytoplasmic proteins were separately extracted from the transduced hMSCs (MOI of 100) and analyzed by Western blotting using primary antibodies specific for human TRIF, phosphorylated IRF-3 (p-IRF-3), phosphorylated NF-κB (p-NF-κB), or β-actin. Alternatively, cells were immunostained with antibodies specific for human IRF-3 or phosphorylated NF-κB for confocal microscopy. For comparison, the cells were left untreated or were treated with poly(I:C) for 24 h for the same analyses. Magnification, ×1,000.
    Figure Legend Snippet: BV transduction activated the TLR3 signaling pathway. (A) Activation of TRIF, IRF-3, and NF-κB. (B) Nuclear translocation of IRF-3 and NF-κB. The nuclear and cytoplasmic proteins were separately extracted from the transduced hMSCs (MOI of 100) and analyzed by Western blotting using primary antibodies specific for human TRIF, phosphorylated IRF-3 (p-IRF-3), phosphorylated NF-κB (p-NF-κB), or β-actin. Alternatively, cells were immunostained with antibodies specific for human IRF-3 or phosphorylated NF-κB for confocal microscopy. For comparison, the cells were left untreated or were treated with poly(I:C) for 24 h for the same analyses. Magnification, ×1,000.

    Techniques Used: Transduction, Activation Assay, Translocation Assay, Western Blot, Confocal Microscopy

    10) Product Images from "Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis"

    Article Title: Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00694-18

    Clathrin-TRIF-dependent NF-κB activation in SL-treated THP-1 cells. (A) Schematic of the experimental design. (B) THP-1 cells were transfected with clathrin siRNA, pretreated with α2,3- or α2,6-SL (10 μM) for 3 h, and then treated with PAK (MOI of 50). IκBα degradation was analyzed by Western blotting, and CXCL8 secretion was analyzed by ELISA. Statistical significance was determined by Student's t test. P values were
    Figure Legend Snippet: Clathrin-TRIF-dependent NF-κB activation in SL-treated THP-1 cells. (A) Schematic of the experimental design. (B) THP-1 cells were transfected with clathrin siRNA, pretreated with α2,3- or α2,6-SL (10 μM) for 3 h, and then treated with PAK (MOI of 50). IκBα degradation was analyzed by Western blotting, and CXCL8 secretion was analyzed by ELISA. Statistical significance was determined by Student's t test. P values were

    Techniques Used: Activation Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    11) Product Images from "The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response"

    Article Title: The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

    Journal: Cell Research

    doi: 10.1038/cr.2016.16

    Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P
    Figure Legend Snippet: Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 10 5 ) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A . (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. * P

    Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activation Assay

    12) Product Images from "Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR"

    Article Title: Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR

    Journal: bioRxiv

    doi: 10.1101/2020.05.18.103333

    LPS- and GC-mediated repression of HIV-1 replication in MDMs requires TLR4, TRIF, and type I IFNs. (A) MDMs (2.5 × 10 5 cells/well) were treated with the TLR2 ligand PAM3CSK4 (100 ng/ml), the TLR4 ligand LPS (100 ng/ml), both PAM3CSK4 and LPS (each 100 ng/ml), or GC (MOI = 10) for 18 hours. Cell supernatant was harvested, filtered through a 0.2 μm filter, and analyzed by ELISA for TNF-α (A) and IFN-β (B) production. Data represent mean (+/− SD) of 3 donors, each donor tested in triplicate. (B) MDMs (2.5 × 10 5 cells/well) were infected with a single-round, replication-competent HIV-luciferase reporter virus at an MOI = 0.1. Forty-eight hours after infection, cells were treated with LPS (100 ng/ml) or GC (MOI = 10) in the absence (white bars) or presence (black bars) of B18R (100 ng/ml) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (C) MDMs (2.5 × 10 5 cells/well) were infected as above. Forty-eight hours after infection, cells were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), or GC (MOI = 10) in the absence (white bars) or presence (black bars) of TAK242 (1 μg/ml) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (D) MDMs (2.5 × 10 5 cells/well) were infected as above. Forty-eight hours after infection, cells were treated with vehicle control (white bars) or with (black bars) the dynamin inhibitor Dynasore (80 μM) for 15 minutes prior to treatment with LPS (100 ng/ml) or GC (MOI = 10) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (E) MDMs (2×10 6 cells/well) were transfected with a control scrambled shRNA, shRNA targeting MyD88, or shRNA targeting TRIF. Knock-down of protein expression was detected by western blot. (F) MDMs (2×10 6 cells/well) were transfected with a control scrambled shRNA (white bars) or with shRNA targeting MyD88 (black bars) or TRIF (gray bars), were infected with a single-round, replication-competent HIV-luciferase reporter virus at an MOI = 0.1. Forty-eight hours after infection, cells were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), both PAM3CSK4 and LPS (100 ng/ml each), GC (MOI = 10), or PMA (10 nM) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate.
    Figure Legend Snippet: LPS- and GC-mediated repression of HIV-1 replication in MDMs requires TLR4, TRIF, and type I IFNs. (A) MDMs (2.5 × 10 5 cells/well) were treated with the TLR2 ligand PAM3CSK4 (100 ng/ml), the TLR4 ligand LPS (100 ng/ml), both PAM3CSK4 and LPS (each 100 ng/ml), or GC (MOI = 10) for 18 hours. Cell supernatant was harvested, filtered through a 0.2 μm filter, and analyzed by ELISA for TNF-α (A) and IFN-β (B) production. Data represent mean (+/− SD) of 3 donors, each donor tested in triplicate. (B) MDMs (2.5 × 10 5 cells/well) were infected with a single-round, replication-competent HIV-luciferase reporter virus at an MOI = 0.1. Forty-eight hours after infection, cells were treated with LPS (100 ng/ml) or GC (MOI = 10) in the absence (white bars) or presence (black bars) of B18R (100 ng/ml) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (C) MDMs (2.5 × 10 5 cells/well) were infected as above. Forty-eight hours after infection, cells were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), or GC (MOI = 10) in the absence (white bars) or presence (black bars) of TAK242 (1 μg/ml) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (D) MDMs (2.5 × 10 5 cells/well) were infected as above. Forty-eight hours after infection, cells were treated with vehicle control (white bars) or with (black bars) the dynamin inhibitor Dynasore (80 μM) for 15 minutes prior to treatment with LPS (100 ng/ml) or GC (MOI = 10) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate. (E) MDMs (2×10 6 cells/well) were transfected with a control scrambled shRNA, shRNA targeting MyD88, or shRNA targeting TRIF. Knock-down of protein expression was detected by western blot. (F) MDMs (2×10 6 cells/well) were transfected with a control scrambled shRNA (white bars) or with shRNA targeting MyD88 (black bars) or TRIF (gray bars), were infected with a single-round, replication-competent HIV-luciferase reporter virus at an MOI = 0.1. Forty-eight hours after infection, cells were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), both PAM3CSK4 and LPS (100 ng/ml each), GC (MOI = 10), or PMA (10 nM) for 18 hours. The cells were then lysed and assayed for luciferase activity. The data are the mean (+/− SD) of 3 donors, each donor tested in triplicate.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Luciferase, Activity Assay, Transfection, shRNA, Expressing, Western Blot

    13) Product Images from "Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death"

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0306-6

    Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.
    Figure Legend Snippet: Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

    Techniques Used: Infection, Western Blot, Transfection, Expressing, Activation Assay, Binding Assay

    TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).
    Figure Legend Snippet: TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

    Techniques Used: Activation Assay, Infection, Western Blot, Transfection, Expressing, Incubation, Staining, Fluorescence, Microscopy

    14) Product Images from "Toll-Like Receptor (TLR) 2 and TLR4 Differentially Regulate Doxorubicin Induced Cardiomyopathy in Mice"

    Article Title: Toll-Like Receptor (TLR) 2 and TLR4 Differentially Regulate Doxorubicin Induced Cardiomyopathy in Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0040763

    The schematic illustrates mechanism for the differential efficacy of TLR2 and TLR4 antagonism on DCM. Inactivation of TLR4 blocks both MyD88-p38 kinase and TRIF-IRF3 pathways, which results in the suppression of autophagy. In contrast, inactivation of TLR2 induces a biphasic regulation on autophagic activity, due to the inhibition of MyD88-p38 kinase and PI3K-AKT-mTOR pathways.
    Figure Legend Snippet: The schematic illustrates mechanism for the differential efficacy of TLR2 and TLR4 antagonism on DCM. Inactivation of TLR4 blocks both MyD88-p38 kinase and TRIF-IRF3 pathways, which results in the suppression of autophagy. In contrast, inactivation of TLR2 induces a biphasic regulation on autophagic activity, due to the inhibition of MyD88-p38 kinase and PI3K-AKT-mTOR pathways.

    Techniques Used: Activity Assay, Inhibition

    15) Product Images from "The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1"

    Article Title: The ORF3 Protein of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced NF-κB Signaling via TRADD and RIP1

    Journal: Scientific Reports

    doi: 10.1038/srep27597

    ORF3 suppresses NF-κB signaling via TRADD and RIP1. ( A ) ORF3 suppresses the phosphorylation of IKKβ and IκBα. ( B ) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. ( C,D ) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.
    Figure Legend Snippet: ORF3 suppresses NF-κB signaling via TRADD and RIP1. ( A ) ORF3 suppresses the phosphorylation of IKKβ and IκBα. ( B ) TLR3 pulls down of key downstream molecules detected by a coimmunoprecipitation assay. ( C,D ) TRADD and TAK1 expression levels are down-regulated in ORF3-pretreated cells compared with GFP-pretreated cells after stimulation with Poly(I:C). In contrast, TRIF, TRAF6 and RIP1 expression levels are increased by this stimulation.

    Techniques Used: Co-Immunoprecipitation Assay, Expressing

    16) Product Images from "Baicalin Protects Keratinocytes From Toll-Like Receptor-4 Mediated DNA Damage and Inflammation Following Ultraviolet Irradiation"

    Article Title: Baicalin Protects Keratinocytes From Toll-Like Receptor-4 Mediated DNA Damage and Inflammation Following Ultraviolet Irradiation

    Journal: Photochemistry and photobiology

    doi: 10.1111/php.12505

    Effect of BA on UVB-induced TLR4 pathway proteins expression in PAM212 cells Cells were treated with either BA or with NAC and exposed to 100 mJ/cm 2 UVB as described in the Materials and Methods section. Western blot analysis was performed to determine the protein expression using TLR4 pathway proteins specific antibodies, including TLR4, MyD88, TRIF, TRAF6, NF-κB, iNOS and COX-2. There was an up-regulation of TLR4 and its downstream targets after UVB exposure (p
    Figure Legend Snippet: Effect of BA on UVB-induced TLR4 pathway proteins expression in PAM212 cells Cells were treated with either BA or with NAC and exposed to 100 mJ/cm 2 UVB as described in the Materials and Methods section. Western blot analysis was performed to determine the protein expression using TLR4 pathway proteins specific antibodies, including TLR4, MyD88, TRIF, TRAF6, NF-κB, iNOS and COX-2. There was an up-regulation of TLR4 and its downstream targets after UVB exposure (p

    Techniques Used: Expressing, Western Blot

    17) Product Images from "Fas-Associated Factor 1 Negatively Regulates the Antiviral Immune Response by Inhibiting Translocation of Interferon Regulatory Factor 3 to the Nucleus"

    Article Title: Fas-Associated Factor 1 Negatively Regulates the Antiviral Immune Response by Inhibiting Translocation of Interferon Regulatory Factor 3 to the Nucleus

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00744-15

    Schematic diagram of FAF1 action on the innate-immunity signaling pathway. Virus infection recruits the kinases TBK1 and IKKε to adaptor proteins MAVS and TRIF. These kinases phosphorylate (p) IRF3, and phosphorylated IRF3 forms dimers which translocate into the nucleus via interaction with IPO5 to produce IFN-β. However, FAF1 inhibits IFN-β activation and ISG induction by interfering with the IRF3-IPO5 interaction and represses nuclear translocation of IRF3.
    Figure Legend Snippet: Schematic diagram of FAF1 action on the innate-immunity signaling pathway. Virus infection recruits the kinases TBK1 and IKKε to adaptor proteins MAVS and TRIF. These kinases phosphorylate (p) IRF3, and phosphorylated IRF3 forms dimers which translocate into the nucleus via interaction with IPO5 to produce IFN-β. However, FAF1 inhibits IFN-β activation and ISG induction by interfering with the IRF3-IPO5 interaction and represses nuclear translocation of IRF3.

    Techniques Used: Infection, Activation Assay, Translocation Assay

    18) Product Images from "Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis"

    Article Title: Bacterial Clearance Is Enhanced by α2,3- and α2,6-Sialyllactose via Receptor-Mediated Endocytosis and Phagocytosis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00694-18

    Clathrin-TRIF-dependent NF-κB activation in SL-treated THP-1 cells. (A) Schematic of the experimental design. (B) THP-1 cells were transfected with clathrin siRNA, pretreated with α2,3- or α2,6-SL (10 μM) for 3 h, and then treated with PAK (MOI of 50). IκBα degradation was analyzed by Western blotting, and CXCL8 secretion was analyzed by ELISA. Statistical significance was determined by Student's t test. P values were
    Figure Legend Snippet: Clathrin-TRIF-dependent NF-κB activation in SL-treated THP-1 cells. (A) Schematic of the experimental design. (B) THP-1 cells were transfected with clathrin siRNA, pretreated with α2,3- or α2,6-SL (10 μM) for 3 h, and then treated with PAK (MOI of 50). IκBα degradation was analyzed by Western blotting, and CXCL8 secretion was analyzed by ELISA. Statistical significance was determined by Student's t test. P values were

    Techniques Used: Activation Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    19) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus-Encoded Replication and Transcription Activator Impairs Innate Immunity via Ubiquitin-Mediated Degradation of Myeloid Differentiation Factor 88"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus-Encoded Replication and Transcription Activator Impairs Innate Immunity via Ubiquitin-Mediated Degradation of Myeloid Differentiation Factor 88

    Journal: Journal of Virology

    doi: 10.1128/JVI.02591-14

    RTA reduces MyD88 expression. (A) One μg pCDH-RTA or vector control was transfected into HeLa cells in 12-well plates. After 36 h, protein levels of MyD88, TRIF, MAVS, IRAK1, IRF3, and IRF7 were determined by Western blotting. GAPDH was the loading
    Figure Legend Snippet: RTA reduces MyD88 expression. (A) One μg pCDH-RTA or vector control was transfected into HeLa cells in 12-well plates. After 36 h, protein levels of MyD88, TRIF, MAVS, IRAK1, IRF3, and IRF7 were determined by Western blotting. GAPDH was the loading

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot

    20) Product Images from "TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling"

    Article Title: TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

    Journal: Yonsei Medical Journal

    doi: 10.3349/ymj.2018.59.1.43

    TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. * p
    Figure Legend Snippet: TRIM56 knockdown inactivated TLR3/TRIF signaling pathway in MM cells. (A) Western blot analysis of TLR3, TRIF, and RIP1 protein in RPMI8226 cells transfected with T56si-2 or si-control. (B) Quantitative analysis of TLR3, TRIF, and RIP1 protein levels. * p

    Techniques Used: Western Blot, Transfection

    TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. * p
    Figure Legend Snippet: TRIM56 deficiency promoted the progression of MM via interrupting TLR3/TRIF signaling pathway. RPMI8226 cells were transfected with T56si-2 or along with poly (I:C) for further analysis at 48 h post-transfection. (A) Cell viability of transfected RPMI8226 cells was assessed by MTT assay. (B) Apoptotic rate of transfected RPMI8226 cells was detected by flow cytometry. (C) The relative caspase3 activity of transfected RPMI8226 cells was measured by the Caspase3 Colorimetric Assay Kit. The concentrations of secreted cytokines IFN-β (D), IL-6 (E), and TNF-α (F) in transfected RPMI8226 cells were measured by ELISA. (G) Western blot analysis of TLR3, TRIF, and RIP1 protein expression in transfected RPMI8226 cells. * p

    Techniques Used: Transfection, MTT Assay, Flow Cytometry, Cytometry, Activity Assay, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

    21) Product Images from "Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy"

    Article Title: Ethanol extract of propolis and its constituent caffeic acid phenethyl ester inhibit breast cancer cells proliferation in inflammatory microenvironment by inhibiting TLR4 signal pathway and inducing apoptosis and autophagy

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-017-1984-9

    EECP and CAPE regulated the levels of TLR4, MyD88, IRAK4 and TRIF. a Cells were treated with EECP and CAPE for 24 h, respectively. Cells were stained with anti-TLR4 antibody. Immunofluorescence graphs showed a decrease of TLR4 level. b The expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells were detected by western blotting at 48 h. c Quantification of relative expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells. ( * P
    Figure Legend Snippet: EECP and CAPE regulated the levels of TLR4, MyD88, IRAK4 and TRIF. a Cells were treated with EECP and CAPE for 24 h, respectively. Cells were stained with anti-TLR4 antibody. Immunofluorescence graphs showed a decrease of TLR4 level. b The expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells were detected by western blotting at 48 h. c Quantification of relative expression of TLR4, MyD88, IRAK4 and TRIF in LPS-stimulated MDA-MB-231 cells. ( * P

    Techniques Used: Staining, Immunofluorescence, Expressing, Multiple Displacement Amplification, Western Blot

    Related Articles

    Transfection:

    Article Title: An alanine to proline mutation in the BB-loop of Toll-like receptor 3 TIR domain switches signalling adaptor specificity from TRIF to MyD88
    Article Snippet: .. Forty eight hours after transfection the cells were either transfected with the relevant constructs or analysed by real-time quantitative PCR (qPCR) or western blot using anti-TRIF (Cell Signaling) and β-actin (Ambion).). .. The cDNA served as template for real-time qPCR analysis to determine the level of gene knockdown using Maxima SYBR Green/ROX qPCR SuperMix (Thermo Scientific).

    Construct:

    Article Title: An alanine to proline mutation in the BB-loop of Toll-like receptor 3 TIR domain switches signalling adaptor specificity from TRIF to MyD88
    Article Snippet: .. Forty eight hours after transfection the cells were either transfected with the relevant constructs or analysed by real-time quantitative PCR (qPCR) or western blot using anti-TRIF (Cell Signaling) and β-actin (Ambion).). .. The cDNA served as template for real-time qPCR analysis to determine the level of gene knockdown using Maxima SYBR Green/ROX qPCR SuperMix (Thermo Scientific).

    Real-time Polymerase Chain Reaction:

    Article Title: An alanine to proline mutation in the BB-loop of Toll-like receptor 3 TIR domain switches signalling adaptor specificity from TRIF to MyD88
    Article Snippet: .. Forty eight hours after transfection the cells were either transfected with the relevant constructs or analysed by real-time quantitative PCR (qPCR) or western blot using anti-TRIF (Cell Signaling) and β-actin (Ambion).). .. The cDNA served as template for real-time qPCR analysis to determine the level of gene knockdown using Maxima SYBR Green/ROX qPCR SuperMix (Thermo Scientific).

    Incubation:

    Article Title: Inflammatory cytokine production in tumor cells upon chemotherapy drug exposure or upon selection for drug resistance
    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). .. All primary antibodies were diluted in TBST supplemented with 5% BSA (Sigma).

    other:

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

    Article Title: The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling
    Article Snippet: Rabbit polyclonal antibodies to TRIF and MAVS (human and rodent specific) were purchased from Cell Signaling Technologies or Bethyl Laboratories, respectively.

    Western Blot:

    Article Title: Type I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors
    Article Snippet: .. Immunoblots were conducted to determine levels of cathepsin E, S, G, K, B, W, Z (all cathepsin antibodies from Santa Cruz Biotechnology, Paso Robles, CA), MDA5, RIG-I, MAVS, Trif, MyD88, p-IRF3(Ser396), IRF3, p-TBK1(Ser172), TBK1/NAK and actin (all remaining antibodies from Cell Signaling Technologies, Danvers, MA). .. Chemiluminescence detection was performed using the Bio-Rad Laboratories Molecular Imager ChemiDoc XRS+ imaging system.

    Article Title: Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection
    Article Snippet: .. Immunoblots were conducted to determine levels of p-IRF3(ser396), IRF3, p-TBK1/NAK(Ser172), TBK1/NAK, RIG-I, MDA5, LGP2, TLR2, TLR3, TLR7, TLR8, TLR9, Trif, MyD88, IRAK1, NOD2 and actin (all antibodies from Cell Signaling Technologies). .. TLRs, NOD1, NOD2, NXLR1, RIG-I, MDA5, Trif and actin expressions were determined by qPCR using Taqman probes (Life technologies/Applied Biosystems).

    Article Title: An alanine to proline mutation in the BB-loop of Toll-like receptor 3 TIR domain switches signalling adaptor specificity from TRIF to MyD88
    Article Snippet: .. Forty eight hours after transfection the cells were either transfected with the relevant constructs or analysed by real-time quantitative PCR (qPCR) or western blot using anti-TRIF (Cell Signaling) and β-actin (Ambion).). .. The cDNA served as template for real-time qPCR analysis to determine the level of gene knockdown using Maxima SYBR Green/ROX qPCR SuperMix (Thermo Scientific).

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    Cell Signaling Technology Inc anti trif antibody
    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 <t>PTEN</t> 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, <t>TRIF</t> 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.
    Anti Trif Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.

    Journal: BMC Immunology

    Article Title: Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection

    doi: 10.1186/s12865-014-0041-4

    Figure Lengend Snippet: Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.

    Article Snippet: Immunoblots were conducted to determine levels of p-IRF3(ser396), IRF3, p-TBK1/NAK(Ser172), TBK1/NAK, RIG-I, MDA5, LGP2, TLR2, TLR3, TLR7, TLR8, TLR9, Trif, MyD88, IRAK1, NOD2 and actin (all antibodies from Cell Signaling Technologies).

    Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Concentration Assay, Infection

    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

    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