rabbit monoclonal antibodies against tlr3  (Cell Signaling Technology Inc)

 
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    Rabbit DA1E mAb IgG XP Isotype Control PE Conjugate
    Description:
    This Cell Signaling Technology antibody is conjugated to phycoerythrin PE and tested in house for direct flow cytometry analysis in human cells
    Catalog Number:
    5742
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    Antibody Conjugates
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    Structured Review

    Cell Signaling Technology Inc rabbit monoclonal antibodies against tlr3
    The RNA-binding activity of Mex3B and proteolytic cleavage of <t>TLR3</t> are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.
    This Cell Signaling Technology antibody is conjugated to phycoerythrin PE and tested in house for direct flow cytometry analysis in human cells
    https://www.bioz.com/result/rabbit monoclonal antibodies against tlr3/product/Cell Signaling Technology Inc
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    1) 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

    The RNA-binding activity of Mex3B and proteolytic cleavage of TLR3 are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.
    Figure Legend Snippet: The RNA-binding activity of Mex3B and proteolytic cleavage of TLR3 are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.

    Techniques Used: RNA Binding Assay, Activity Assay, Expressing, Transfection, Luciferase

    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

    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P
    Figure Legend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.
    Figure Legend Snippet: Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

    Techniques Used: Ligand Binding Assay, Marker, Transfection, Staining, Confocal Microscopy, Isolation, Over Expression, Plasmid Preparation, Binding Assay, Incubation

    A working model on the involvement of Mex3B in TLR3-mediated signaling. Upon viral infection, TLR3 transports from the ER to endosomes where it is proteolytically cleaved, resulting in the formation of a very stable “cleaved/associated” TLR3 that represents the primary form of the signaling receptor. The RNA-binding protein Mex3B localizes in the lumen of endosomes, functions as a coreceptor of TLR3 by promoting ligand binding and proteolytic processing of TLR3, resulting in full activation of TLR3.
    Figure Legend Snippet: A working model on the involvement of Mex3B in TLR3-mediated signaling. Upon viral infection, TLR3 transports from the ER to endosomes where it is proteolytically cleaved, resulting in the formation of a very stable “cleaved/associated” TLR3 that represents the primary form of the signaling receptor. The RNA-binding protein Mex3B localizes in the lumen of endosomes, functions as a coreceptor of TLR3 by promoting ligand binding and proteolytic processing of TLR3, resulting in full activation of TLR3.

    Techniques Used: Infection, RNA Binding Assay, Ligand Binding Assay, Activation Assay

    Mex3B is a positive regulator of TLR3-mediated signaling. (A) Mex3B potentiates TLR3-mediated signaling in 293 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 6 h before luciferase assays. (B) Mex3B potentiates poly(I:C)-induced signaling in 293-TLR3 cells. Reporter assays were similarly performed as in A . (C) Mex3B potentiates poly(I:C)-induced expression of downstream genes in 293-TLR3 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 3 h before qPCR analysis. (D) Mex3B potentiates poly(I:C)-induced activation of the IFN-β promoter in HeLa and HCT116 cells. Reporter assays were similarly performed as in A . (E) Overexpression of Mex3B has no marked effects on SeV- or cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. The 293 cells were transfected with the indicated plasmids for 24 h, and then infected with SeV or re-transfected with poly(I:C) for 12 h before luciferase assays. (F) Effects of Mex3B-RNAi on expression of Mex3B. The 293 cells were transfected with a Mex3B-RNAi plasmid for 24 h before immunoblot analysis. (G) Effects of Mex3B-RNAi on TLR3-mediated signaling. Reporter assays were similarly performed as in A . (H) Effects of Mex3B-RNAi on poly(I:C)-induced transcription of IFNB1 , ISG56 and TNFA genes. The experiments were similarly performed as in C . (I) Effects of Mex3B-RNAi on poly(I:C)- and SeV-induced activation of the IFN-β promoter in HCT116 cells. The cells were transfected with the indicated plasmids for 24 h, and then infected with SeV for 12 h or treated with poly(I:C) for 6 h before luciferase assays. Data are represented as mean ± SEM. * P
    Figure Legend Snippet: Mex3B is a positive regulator of TLR3-mediated signaling. (A) Mex3B potentiates TLR3-mediated signaling in 293 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 6 h before luciferase assays. (B) Mex3B potentiates poly(I:C)-induced signaling in 293-TLR3 cells. Reporter assays were similarly performed as in A . (C) Mex3B potentiates poly(I:C)-induced expression of downstream genes in 293-TLR3 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 3 h before qPCR analysis. (D) Mex3B potentiates poly(I:C)-induced activation of the IFN-β promoter in HeLa and HCT116 cells. Reporter assays were similarly performed as in A . (E) Overexpression of Mex3B has no marked effects on SeV- or cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. The 293 cells were transfected with the indicated plasmids for 24 h, and then infected with SeV or re-transfected with poly(I:C) for 12 h before luciferase assays. (F) Effects of Mex3B-RNAi on expression of Mex3B. The 293 cells were transfected with a Mex3B-RNAi plasmid for 24 h before immunoblot analysis. (G) Effects of Mex3B-RNAi on TLR3-mediated signaling. Reporter assays were similarly performed as in A . (H) Effects of Mex3B-RNAi on poly(I:C)-induced transcription of IFNB1 , ISG56 and TNFA genes. The experiments were similarly performed as in C . (I) Effects of Mex3B-RNAi on poly(I:C)- and SeV-induced activation of the IFN-β promoter in HCT116 cells. The cells were transfected with the indicated plasmids for 24 h, and then infected with SeV for 12 h or treated with poly(I:C) for 6 h before luciferase assays. Data are represented as mean ± SEM. * P

    Techniques Used: Transfection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Over Expression, Infection, Plasmid Preparation

    Mex3B is associated with TLR3 and facilitates the recruitment of TRIF upon poly(I:C) stimulation. (A) Mex3B interacts with TLR3. HCT116(Mex3B-3×Flag) cells (1 × 10 8 ), in which a cDNA encoding three copies of Flag tag was inserted immediately before the stop codon of Mex3b gene 32 , or mouse DCs (1 × 10 8 ) were treated with poly(I:C) (20 μg /ml) or LPS (20 ng/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. pIC, poly(I:C). (B) Domain mapping of the Mex3B-TLR3 interaction. The 293-TLR3 cells (2 × 10 6 ) were transfected with either the full-length or the indicated Mex3B truncation plasmids (5 μg). Coimmunoprecipitation and immunoblot were performed with the indicated antibodies. Ig, mouse IgG; αT3, anti-TLR3. (C) Mex3B interacts with TRIF upon poly(I:C) stimulation. HCT116 (Mex3B-3×Flag) cells (1 × 10 8 ) were left untreated or treated with poly(I:C) (20 μg/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies.
    Figure Legend Snippet: Mex3B is associated with TLR3 and facilitates the recruitment of TRIF upon poly(I:C) stimulation. (A) Mex3B interacts with TLR3. HCT116(Mex3B-3×Flag) cells (1 × 10 8 ), in which a cDNA encoding three copies of Flag tag was inserted immediately before the stop codon of Mex3b gene 32 , or mouse DCs (1 × 10 8 ) were treated with poly(I:C) (20 μg /ml) or LPS (20 ng/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. pIC, poly(I:C). (B) Domain mapping of the Mex3B-TLR3 interaction. The 293-TLR3 cells (2 × 10 6 ) were transfected with either the full-length or the indicated Mex3B truncation plasmids (5 μg). Coimmunoprecipitation and immunoblot were performed with the indicated antibodies. Ig, mouse IgG; αT3, anti-TLR3. (C) Mex3B interacts with TRIF upon poly(I:C) stimulation. HCT116 (Mex3B-3×Flag) cells (1 × 10 8 ) were left untreated or treated with poly(I:C) (20 μg/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies.

    Techniques Used: FLAG-tag, Western Blot, Transfection

    2) Product Images from "ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3"

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mjaa004

    Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P
    Figure Legend Snippet: Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P

    Techniques Used: In Vivo, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Zcchc3-deficiency impairs TLR3-mediated signaling in primary mouse cells. ( A and B ) Effects of Zcchc3-deficiency on poly(I:C)- or LPS-induced transcription of Ifnb1 , Isg56 , and Il6 in BMDMs ( A ) and BMDCs ( B ). Zcchc3 +/+ and Zcchc3 −/− cells (3 × 10 5 ) were treated with poly(I:C) (20 μg/ml) or LPS (50 ng/ml) for the indicated times before qPCR was performed (* P
    Figure Legend Snippet: Zcchc3-deficiency impairs TLR3-mediated signaling in primary mouse cells. ( A and B ) Effects of Zcchc3-deficiency on poly(I:C)- or LPS-induced transcription of Ifnb1 , Isg56 , and Il6 in BMDMs ( A ) and BMDCs ( B ). Zcchc3 +/+ and Zcchc3 −/− cells (3 × 10 5 ) were treated with poly(I:C) (20 μg/ml) or LPS (50 ng/ml) for the indicated times before qPCR was performed (* P

    Techniques Used: Real-time Polymerase Chain Reaction

    ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.
    Figure Legend Snippet: ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

    Techniques Used: Over Expression, Binding Assay, Transfection, Incubation, Isolation, Immunoprecipitation

    ZCCHC3 is associated with TLR3 and TRIF. ( A – C ) HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting were performed with anti-HA-peroxidase and anti-FLAG-M2-peroxidase. ( A ) ZCCHC3 is associated with TLR3 and TRIF. ( B ) Interaction of ZCCHC3 with TLR3 mutants and TRIF mutants. ( C ) Interaction of ZCCHC3 mutants with TLR3 and TRIF. ( D ) Endogenous ZCCHC3 is associated with TLR3 and TRIF in HT1080 cells. HT1080 cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting.
    Figure Legend Snippet: ZCCHC3 is associated with TLR3 and TRIF. ( A – C ) HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting were performed with anti-HA-peroxidase and anti-FLAG-M2-peroxidase. ( A ) ZCCHC3 is associated with TLR3 and TRIF. ( B ) Interaction of ZCCHC3 with TLR3 mutants and TRIF mutants. ( C ) Interaction of ZCCHC3 mutants with TLR3 and TRIF. ( D ) Endogenous ZCCHC3 is associated with TLR3 and TRIF in HT1080 cells. HT1080 cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting.

    Techniques Used: Transfection, Immunoprecipitation

    ZCCHC3 positively regulates poly(I:C)-triggered signaling. ( A ) ZCCHC3 promotes poly(I:C)-induced activation of the IFN-β promoter and ISRE in a dose-dependent manner. 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter plasmids (0.1 μg) and increased amounts of ZCCHC3 plasmids (5, 10, 20 ng) for 18 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays. ( B ) Efficiencies of ZCCHC3-RNAi plasmids on ZCCHC3 levels. 293 cells (4 × 10 5 ) were transfected with ZCCHC3-RNAi and control RNAi plasmids for 24 h before immunoblotting. ( C ) Effects of ZCCHC3 knockdown on poly(I:C)-induced activation of the IFN-β promoter and ISRE. 293-TLR3 (1 × 10 5 ) cells were transfected with the indicated luciferase reporter and RNAi plasmids. Aftre 20 h, cells were treated with poly(I:C) (20 μg/ml) or left untreated for 6 h before luciferase assays (** P
    Figure Legend Snippet: ZCCHC3 positively regulates poly(I:C)-triggered signaling. ( A ) ZCCHC3 promotes poly(I:C)-induced activation of the IFN-β promoter and ISRE in a dose-dependent manner. 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter plasmids (0.1 μg) and increased amounts of ZCCHC3 plasmids (5, 10, 20 ng) for 18 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays. ( B ) Efficiencies of ZCCHC3-RNAi plasmids on ZCCHC3 levels. 293 cells (4 × 10 5 ) were transfected with ZCCHC3-RNAi and control RNAi plasmids for 24 h before immunoblotting. ( C ) Effects of ZCCHC3 knockdown on poly(I:C)-induced activation of the IFN-β promoter and ISRE. 293-TLR3 (1 × 10 5 ) cells were transfected with the indicated luciferase reporter and RNAi plasmids. Aftre 20 h, cells were treated with poly(I:C) (20 μg/ml) or left untreated for 6 h before luciferase assays (** P

    Techniques Used: Activation Assay, Transfection, Luciferase

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    Article Snippet: .. Materials (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl) butanamide (CFMB) and TNF-α antagonist R-7050 [ ] (Calbiochem, Darmstadt, Germany); sodium propionate (NaP) (Sigma-Aldrich, St. Louis, MO, USA); pertussis toxin (PTX) (Wako, Osaka, Japan); gallein, cyclopropanecarboxylic acid (CPC) and trichostatin A (TSA) (Tokyo Chemical Industry, Tokyo, Japan); human recombinant TNF-α (PeproTech, Rocky Hill, NJ, USA); polyclonal rabbit antibodies against human β-actin (Abcam), acetyl-histone H3 (Lys9/Lys14), cleaved caspase-3 (Asp175), cleaved caspase-9 (all Cell Signaling Technology, Boston, MA USA), GPR41 (Abcam), and GPR43 (Bioss, Boston, MA, USA); monoclonal rabbit antibodies against HDAC1, HDAC4, HDAC5 (Cell Signaling Technology) and HDAC8 (Abcam); monoclonal mouse antibodies against HDAC3 and caspase-8 (Cell Signaling Technology); and horseradish peroxidase-conjugated anti-mouse, anti-goat and anti-rabbit immunoglobulins (Dako, Glostrup, Denmark) were used in the study. .. Cell cultures HepG2 cells, HuH-7 cells, JHH-4 cells and HLE cells, which are all human hepatoma cell lines, were obtained from the Japanese Collection of Research Bioresources Cell Bank.

    other:

    Article Title: FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis
    Article Snippet: Antibodies Rabbit antiserum against FoxO3 (2497), cleaved caspase 3 (C9661), and cleaved poly (ADP-ribose) polymerase (PARP) (P9542) were from Cell Signaling.

    SDS Page:

    Article Title: Germinal Center hypoxia and regulation of antibody qualities by a hypoxia response system
    Article Snippet: .. Proteins in whole cell extracts were separated by SDS-PAGE, transferred onto nylon membranes (Millipore), and then incubated with rabbit antibodies against p-S6 (S235/236), p-p70S6K (S371), p-Akt (S473), p-Akt (T308), p-ACC (S79), p-AMPK (T172) (Cell Signaling Technologies), or goat anti-Actin (Santa Cruz) Abs followed by the appropriate fluorophore-conjugated, species-specific secondary anti-Ig antibodies (Rockland Immunochemicals, and LI-COR). .. Proteins were visualized and quantitated by laser excitation and infrared imaging (Odyssey, LI-COR).

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    Cell Signaling Technology Inc rabbit monoclonal antibodies against tlr3
    The RNA-binding activity of Mex3B and proteolytic cleavage of <t>TLR3</t> are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.
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    The RNA-binding activity of Mex3B and proteolytic cleavage of TLR3 are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: The RNA-binding activity of Mex3B and proteolytic cleavage of TLR3 are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B - D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 10 5 ) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: RNA Binding Assay, Activity Assay, Expressing, Transfection, Luciferase

    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

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend 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

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activation Assay

    Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 10 5 ) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C , D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D) . The cells (2 × 10 5 ) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 10 5 ) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 10 5 ) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 10 5 ) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Ligand Binding Assay, Marker, Transfection, Staining, Confocal Microscopy, Isolation, Over Expression, Plasmid Preparation, Binding Assay, Incubation

    A working model on the involvement of Mex3B in TLR3-mediated signaling. Upon viral infection, TLR3 transports from the ER to endosomes where it is proteolytically cleaved, resulting in the formation of a very stable “cleaved/associated” TLR3 that represents the primary form of the signaling receptor. The RNA-binding protein Mex3B localizes in the lumen of endosomes, functions as a coreceptor of TLR3 by promoting ligand binding and proteolytic processing of TLR3, resulting in full activation of TLR3.

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: A working model on the involvement of Mex3B in TLR3-mediated signaling. Upon viral infection, TLR3 transports from the ER to endosomes where it is proteolytically cleaved, resulting in the formation of a very stable “cleaved/associated” TLR3 that represents the primary form of the signaling receptor. The RNA-binding protein Mex3B localizes in the lumen of endosomes, functions as a coreceptor of TLR3 by promoting ligand binding and proteolytic processing of TLR3, resulting in full activation of TLR3.

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Infection, RNA Binding Assay, Ligand Binding Assay, Activation Assay

    Mex3B is a positive regulator of TLR3-mediated signaling. (A) Mex3B potentiates TLR3-mediated signaling in 293 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 6 h before luciferase assays. (B) Mex3B potentiates poly(I:C)-induced signaling in 293-TLR3 cells. Reporter assays were similarly performed as in A . (C) Mex3B potentiates poly(I:C)-induced expression of downstream genes in 293-TLR3 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 3 h before qPCR analysis. (D) Mex3B potentiates poly(I:C)-induced activation of the IFN-β promoter in HeLa and HCT116 cells. Reporter assays were similarly performed as in A . (E) Overexpression of Mex3B has no marked effects on SeV- or cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. The 293 cells were transfected with the indicated plasmids for 24 h, and then infected with SeV or re-transfected with poly(I:C) for 12 h before luciferase assays. (F) Effects of Mex3B-RNAi on expression of Mex3B. The 293 cells were transfected with a Mex3B-RNAi plasmid for 24 h before immunoblot analysis. (G) Effects of Mex3B-RNAi on TLR3-mediated signaling. Reporter assays were similarly performed as in A . (H) Effects of Mex3B-RNAi on poly(I:C)-induced transcription of IFNB1 , ISG56 and TNFA genes. The experiments were similarly performed as in C . (I) Effects of Mex3B-RNAi on poly(I:C)- and SeV-induced activation of the IFN-β promoter in HCT116 cells. The cells were transfected with the indicated plasmids for 24 h, and then infected with SeV for 12 h or treated with poly(I:C) for 6 h before luciferase assays. Data are represented as mean ± SEM. * P

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is a positive regulator of TLR3-mediated signaling. (A) Mex3B potentiates TLR3-mediated signaling in 293 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 6 h before luciferase assays. (B) Mex3B potentiates poly(I:C)-induced signaling in 293-TLR3 cells. Reporter assays were similarly performed as in A . (C) Mex3B potentiates poly(I:C)-induced expression of downstream genes in 293-TLR3 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 3 h before qPCR analysis. (D) Mex3B potentiates poly(I:C)-induced activation of the IFN-β promoter in HeLa and HCT116 cells. Reporter assays were similarly performed as in A . (E) Overexpression of Mex3B has no marked effects on SeV- or cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. The 293 cells were transfected with the indicated plasmids for 24 h, and then infected with SeV or re-transfected with poly(I:C) for 12 h before luciferase assays. (F) Effects of Mex3B-RNAi on expression of Mex3B. The 293 cells were transfected with a Mex3B-RNAi plasmid for 24 h before immunoblot analysis. (G) Effects of Mex3B-RNAi on TLR3-mediated signaling. Reporter assays were similarly performed as in A . (H) Effects of Mex3B-RNAi on poly(I:C)-induced transcription of IFNB1 , ISG56 and TNFA genes. The experiments were similarly performed as in C . (I) Effects of Mex3B-RNAi on poly(I:C)- and SeV-induced activation of the IFN-β promoter in HCT116 cells. The cells were transfected with the indicated plasmids for 24 h, and then infected with SeV for 12 h or treated with poly(I:C) for 6 h before luciferase assays. Data are represented as mean ± SEM. * P

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: Transfection, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Activation Assay, Over Expression, Infection, Plasmid Preparation

    Mex3B is associated with TLR3 and facilitates the recruitment of TRIF upon poly(I:C) stimulation. (A) Mex3B interacts with TLR3. HCT116(Mex3B-3×Flag) cells (1 × 10 8 ), in which a cDNA encoding three copies of Flag tag was inserted immediately before the stop codon of Mex3b gene 32 , or mouse DCs (1 × 10 8 ) were treated with poly(I:C) (20 μg /ml) or LPS (20 ng/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. pIC, poly(I:C). (B) Domain mapping of the Mex3B-TLR3 interaction. The 293-TLR3 cells (2 × 10 6 ) were transfected with either the full-length or the indicated Mex3B truncation plasmids (5 μg). Coimmunoprecipitation and immunoblot were performed with the indicated antibodies. Ig, mouse IgG; αT3, anti-TLR3. (C) Mex3B interacts with TRIF upon poly(I:C) stimulation. HCT116 (Mex3B-3×Flag) cells (1 × 10 8 ) were left untreated or treated with poly(I:C) (20 μg/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies.

    Journal: Cell Research

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

    doi: 10.1038/cr.2016.16

    Figure Lengend Snippet: Mex3B is associated with TLR3 and facilitates the recruitment of TRIF upon poly(I:C) stimulation. (A) Mex3B interacts with TLR3. HCT116(Mex3B-3×Flag) cells (1 × 10 8 ), in which a cDNA encoding three copies of Flag tag was inserted immediately before the stop codon of Mex3b gene 32 , or mouse DCs (1 × 10 8 ) were treated with poly(I:C) (20 μg /ml) or LPS (20 ng/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. pIC, poly(I:C). (B) Domain mapping of the Mex3B-TLR3 interaction. The 293-TLR3 cells (2 × 10 6 ) were transfected with either the full-length or the indicated Mex3B truncation plasmids (5 μg). Coimmunoprecipitation and immunoblot were performed with the indicated antibodies. Ig, mouse IgG; αT3, anti-TLR3. (C) Mex3B interacts with TRIF upon poly(I:C) stimulation. HCT116 (Mex3B-3×Flag) cells (1 × 10 8 ) were left untreated or treated with poly(I:C) (20 μg/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies.

    Article Snippet: Reagents and antibodies Poly(I:C), LPS, R837, PGN, chloroquine (Invivogen); TNFα, IL-1β (R & D Systems); D-galactosamine (Sigma); GM-CSF (peproTech); ELISA kit for murine IFN-β, TNFα, IL-6 (Biolegend); dual-specific luciferase assay kit (Promega); EZ-link Psoralen-PEG3 -Biotin (Thermo); streptavidin agarose (Solulink); lysosome isolation kit (sigma); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (OriGene), phospho-IBα (Cell Signaling Technology), TLR3 (IMGENEX); rabbit monoclonal antibodies against TLR3, phospho-IRF3 (Cell Signaling Technology); rabbit polyclonal antibodies against IRF3 (Santa Cruz Biotechnology), TRIF (Cell Signaling Technology), TBK1 and phospho-TBK1(EPITOMICS), Flag (MBL); Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, Alexa Fluor 647-conjugated goat anti-mouse IgG antibody (Invitrogen) were purchased from the indicated manufacturers.

    Techniques: FLAG-tag, Western Blot, Transfection

    Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: Zcchc3-deficiency impairs TLR3-mediated immune responses in vivo . ( A ) Serum cytokine concentrations in Zcchc3 +/+ and Zcchc3 −/− mice. Sex- and age-matched Zcchc3 +/+ and Zcchc3 −/− mice ( n = 6 or 7) were infected with poly(I:C) (2 μg/g) plus D-galactosamine (1 mg/g) or LPS (10 μg/g) for 4 h, and the concentrations of IFN-β, IL-6, and TNFα in the serum were determined by ELISA (*** P

    Article Snippet: Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

    Techniques: In Vivo, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay

    Zcchc3-deficiency impairs TLR3-mediated signaling in primary mouse cells. ( A and B ) Effects of Zcchc3-deficiency on poly(I:C)- or LPS-induced transcription of Ifnb1 , Isg56 , and Il6 in BMDMs ( A ) and BMDCs ( B ). Zcchc3 +/+ and Zcchc3 −/− cells (3 × 10 5 ) were treated with poly(I:C) (20 μg/ml) or LPS (50 ng/ml) for the indicated times before qPCR was performed (* P

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: Zcchc3-deficiency impairs TLR3-mediated signaling in primary mouse cells. ( A and B ) Effects of Zcchc3-deficiency on poly(I:C)- or LPS-induced transcription of Ifnb1 , Isg56 , and Il6 in BMDMs ( A ) and BMDCs ( B ). Zcchc3 +/+ and Zcchc3 −/− cells (3 × 10 5 ) were treated with poly(I:C) (20 μg/ml) or LPS (50 ng/ml) for the indicated times before qPCR was performed (* P

    Article Snippet: Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

    Techniques: Real-time Polymerase Chain Reaction

    ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: ZCCHC3 promotes the recruitment of TRIF to TLR3. ( A ) Overexpression of ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin-sepharose beads. Precipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. ( B ) Endogenous ZCCHC3 has no effects on the binding of TLR3 to poly(I:C). Lysate of 293-TLR3 cells (1 × 10 7 ) was incubated with the indicated biotinylated-poly(I:C) for 1 h before pull-down assays and immunoblotting. ( C ) ZCCHC3 localizes in the cytosolic but not the lumen of endosomes. 293-TLR3 cells (4 × 10 8 ) were collected and washed twice with ice-cold PBS. Endosomal and lysosomal fractions were isolated and left untreated or treated with proteinase K (2 μg/ml) or proteinase K plus 0.2% Triton X-100 for 15 min at room temperature. The samples were then analyzed by immunoblotting with the indicates antibodies. ( D ) ZCCHC3 promotes TLR3–TRIF but not TLR4–TRIF interaction in mammalian overexpression system. HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cells were lysed for co-immunoprecipitation and immunoblotting with the indicated antibodies. ( E ) Knockdown of ZCCHC3 disrupts TLR3–TRIF but not TLR4–TRIF interaction. ZCCHC3-knockdown or control 293-TLR3 cells (2 × 10 6 ) were transfected with the indicated plasmids. After 20 h, the cell lysates were harvested and lysed for co-immunoprecipitation before immunoblotting with the indicated antibodies. ( F ) Knockdown of ZCCHC3 disrupts the recruitment of TRIF to TLR3. ZCCHC3-knockdown or control 293-TLR3 cells (1 × 10 7 ) were treated with poly(I:C) (50 μg/ml) for the indicated times. The cell lysates were immunoprecipitated with anti-TLR3 and analyzed by immunoblotting with the indicated antibodies. ( G ) Stimulation of poly(I:C) but not LPS induces the interaction of ZCCHC3 with TRIF. The indicated cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) or LPS (200 ng/ml) for the indicated times before co-immunoprecipitation and immunoblotting with the indicated antibodies.

    Article Snippet: Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

    Techniques: Over Expression, Binding Assay, Transfection, Incubation, Isolation, Immunoprecipitation

    ZCCHC3 is associated with TLR3 and TRIF. ( A – C ) HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting were performed with anti-HA-peroxidase and anti-FLAG-M2-peroxidase. ( A ) ZCCHC3 is associated with TLR3 and TRIF. ( B ) Interaction of ZCCHC3 with TLR3 mutants and TRIF mutants. ( C ) Interaction of ZCCHC3 mutants with TLR3 and TRIF. ( D ) Endogenous ZCCHC3 is associated with TLR3 and TRIF in HT1080 cells. HT1080 cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting.

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: ZCCHC3 is associated with TLR3 and TRIF. ( A – C ) HEK293 cells (2 × 10 6 ) were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting were performed with anti-HA-peroxidase and anti-FLAG-M2-peroxidase. ( A ) ZCCHC3 is associated with TLR3 and TRIF. ( B ) Interaction of ZCCHC3 with TLR3 mutants and TRIF mutants. ( C ) Interaction of ZCCHC3 mutants with TLR3 and TRIF. ( D ) Endogenous ZCCHC3 is associated with TLR3 and TRIF in HT1080 cells. HT1080 cells (1 × 10 7 ) were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting.

    Article Snippet: Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

    Techniques: Transfection, Immunoprecipitation

    ZCCHC3 positively regulates poly(I:C)-triggered signaling. ( A ) ZCCHC3 promotes poly(I:C)-induced activation of the IFN-β promoter and ISRE in a dose-dependent manner. 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter plasmids (0.1 μg) and increased amounts of ZCCHC3 plasmids (5, 10, 20 ng) for 18 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays. ( B ) Efficiencies of ZCCHC3-RNAi plasmids on ZCCHC3 levels. 293 cells (4 × 10 5 ) were transfected with ZCCHC3-RNAi and control RNAi plasmids for 24 h before immunoblotting. ( C ) Effects of ZCCHC3 knockdown on poly(I:C)-induced activation of the IFN-β promoter and ISRE. 293-TLR3 (1 × 10 5 ) cells were transfected with the indicated luciferase reporter and RNAi plasmids. Aftre 20 h, cells were treated with poly(I:C) (20 μg/ml) or left untreated for 6 h before luciferase assays (** P

    Journal: Journal of Molecular Cell Biology

    Article Title: ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3

    doi: 10.1093/jmcb/mjaa004

    Figure Lengend Snippet: ZCCHC3 positively regulates poly(I:C)-triggered signaling. ( A ) ZCCHC3 promotes poly(I:C)-induced activation of the IFN-β promoter and ISRE in a dose-dependent manner. 293-TLR3 cells (1 × 10 5 ) were transfected with the IFN-β promoter or ISRE reporter plasmids (0.1 μg) and increased amounts of ZCCHC3 plasmids (5, 10, 20 ng) for 18 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays. ( B ) Efficiencies of ZCCHC3-RNAi plasmids on ZCCHC3 levels. 293 cells (4 × 10 5 ) were transfected with ZCCHC3-RNAi and control RNAi plasmids for 24 h before immunoblotting. ( C ) Effects of ZCCHC3 knockdown on poly(I:C)-induced activation of the IFN-β promoter and ISRE. 293-TLR3 (1 × 10 5 ) cells were transfected with the indicated luciferase reporter and RNAi plasmids. Aftre 20 h, cells were treated with poly(I:C) (20 μg/ml) or left untreated for 6 h before luciferase assays (** P

    Article Snippet: Mouse monoclonal antibodies against HA (Origene), FLAG, and β-actin, anti-HA-Peroxidase, and anti-FLAG-M2-Peroxidase (Sigma) and rabbit monoclonal antibodies against TLR3, phosphor-IRF3, p65 (Cell Signaling Technology), phosphor-TBK1, TBK1, TRIF (Abcam), and IRF3 (Santa Cruz Biotechnology) were purchased from the indicated manufacturers.

    Techniques: Activation Assay, Transfection, Luciferase