tlr4  (Sino Biological)


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    Name:
    Human TLR4 qPCR Primer Pair
    Description:
    Verified forward and reverse primers for analyzing the quantitative expression of gene
    Catalog Number:
    HP100217
    Price:
    79.0
    Category:
    qPCR primer pairs
    Reactivity:
    Human
    Size:
    1Unit
    Product Aliases:
    ARMD10 qPCR Primer Pairs Human, CD284 qPCR Primer Pairs Human, TLR-4 qPCR Primer Pairs Human, TLR4 qPCR Primer Pairs Human, TOLL qPCR Primer Pairs Human
    Molecule Name:
    TLR4
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    Structured Review

    Sino Biological tlr4
    DENV NS1 binds to platelets and induces activation through <t>TLR4</t> signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P
    Verified forward and reverse primers for analyzing the quantitative expression of gene
    https://www.bioz.com/result/tlr4/product/Sino Biological
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tlr4 - by Bioz Stars, 2021-09
    80/100 stars

    Images

    1) Product Images from "Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage"

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007625

    DENV NS1 binds to platelets and induces activation through TLR4 signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P
    Figure Legend Snippet: DENV NS1 binds to platelets and induces activation through TLR4 signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P

    Techniques Used: Activation Assay, Transduction, Expressing, Isolation, Western Blot, Binding Assay, Flow Cytometry, Knock-Out, Mouse Assay, Fluorescence

    TLR4 is involved in DENV-induced prolongation of bleeding time and hemorrhage in mice. A hemorrhagic mouse model was performed with C57BL/6J mice (WT) and TLR4 -/- C57BL/6J background mice as described in the Methods. (A) Mouse skin samples were removed to observe local hemorrhage on day 3 after DENV injection. The number of mice with hemorrhage divided by the total number of mice inoculated in each group is indicated. Yellow arrows indicate local skin hemorrhage. (B) The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity. (C) The tail bleeding time and (D) platelet counts were also determined on day 3 before sacrifice. *P
    Figure Legend Snippet: TLR4 is involved in DENV-induced prolongation of bleeding time and hemorrhage in mice. A hemorrhagic mouse model was performed with C57BL/6J mice (WT) and TLR4 -/- C57BL/6J background mice as described in the Methods. (A) Mouse skin samples were removed to observe local hemorrhage on day 3 after DENV injection. The number of mice with hemorrhage divided by the total number of mice inoculated in each group is indicated. Yellow arrows indicate local skin hemorrhage. (B) The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity. (C) The tail bleeding time and (D) platelet counts were also determined on day 3 before sacrifice. *P

    Techniques Used: Mouse Assay, Injection

    Proposed mechanisms of the contribution of DENV NS1 to cause thrombocytopenia and hemorrhage during DENV infection. Circulating DENV NS1 binds to platelets via TLR4 or other molecules to induce the release of ADP, which in turn elevates P-selectin expression and PS exposure on platelet surfaces leading to platelet activation and enhancement of the platelet aggregation. In addition, NS1-activated platelets are prone to adhere onto endothelium or phagocytosis by macrophages. On the other hand, NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release. All these effects induced by NS1 can contribute to the thrombocytopenia and hemorrhage during DENV infection.
    Figure Legend Snippet: Proposed mechanisms of the contribution of DENV NS1 to cause thrombocytopenia and hemorrhage during DENV infection. Circulating DENV NS1 binds to platelets via TLR4 or other molecules to induce the release of ADP, which in turn elevates P-selectin expression and PS exposure on platelet surfaces leading to platelet activation and enhancement of the platelet aggregation. In addition, NS1-activated platelets are prone to adhere onto endothelium or phagocytosis by macrophages. On the other hand, NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release. All these effects induced by NS1 can contribute to the thrombocytopenia and hemorrhage during DENV infection.

    Techniques Used: Infection, Expressing, Activation Assay

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage
    Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA) To examine the direct interaction between DENV NS1 and candidate receptors, 50 μl of TLR4 (purified from baculovirus-system, Sino Biological), TLR2 (purified from baculovirus-system, Sino Biological), His-taq protein or BSA (5 μg/ml) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4 °C overnight. ..

    Purification:

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage
    Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA) To examine the direct interaction between DENV NS1 and candidate receptors, 50 μl of TLR4 (purified from baculovirus-system, Sino Biological), TLR2 (purified from baculovirus-system, Sino Biological), His-taq protein or BSA (5 μg/ml) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4 °C overnight. ..

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    Sino Biological recombinant tlr4 protein
    DC activating effect of API5 is <t>TLR4</t> dependent. Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein or LPS (100ng/ml). (A) Supernatants were collected and levels of various cytokines were assessed using ELISA. (B) Expression of various maturation markers (CD40, CD80, CD86 and MHCI) by treated cells was analyzed using flow cytometry. (C) Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein for 0, 10, 30, or 60 minutes, lysed, and assessed for the expression of various MAPk and IkB-α using western blot analysis. (D) Mice (5 mice per group) were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. One week after second vaccination, splenocytes of mice were collected and assessed for E7-specific CD8+ T cells. (E-F) Mice (5 mice per group) were challenged with TC-1 cells. Three days after tumor challenge, mice were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. (E) Tumor growth was measured using digital caliper. (F) Mice survival were assessed using Kaplan-Meier analysis. Data are presented as mean ± SD (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).
    Recombinant Tlr4 Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant tlr4 protein/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant tlr4 protein - by Bioz Stars, 2021-09
    94/100 stars
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    80
    Sino Biological tlr4
    DENV NS1 binds to platelets and induces activation through <t>TLR4</t> signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P
    Tlr4, supplied by Sino Biological, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tlr4/product/Sino Biological
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tlr4 - by Bioz Stars, 2021-09
    80/100 stars
      Buy from Supplier

    94
    Sino Biological anti toll like receptor 4 tlr4 antibody
    Effects of Danshensu on the protein expression of p-NF-κB p65 and <t>TLR4</t> in SARS-CoV-2 S-induced lung tissues and the mRNA expression of ACE2 and AGT. a Western blotting of proteins involved in TLR4, NF-κB p65 and p-NF-κB p65. b The mRNA level of ACE2. c The mRNA level of AGT. Compared to SARS-CoV-2 S group, * P
    Anti Toll Like Receptor 4 Tlr4 Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti toll like receptor 4 tlr4 antibody/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti toll like receptor 4 tlr4 antibody - by Bioz Stars, 2021-09
    94/100 stars
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    Image Search Results


    DC activating effect of API5 is TLR4 dependent. Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein or LPS (100ng/ml). (A) Supernatants were collected and levels of various cytokines were assessed using ELISA. (B) Expression of various maturation markers (CD40, CD80, CD86 and MHCI) by treated cells was analyzed using flow cytometry. (C) Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein for 0, 10, 30, or 60 minutes, lysed, and assessed for the expression of various MAPk and IkB-α using western blot analysis. (D) Mice (5 mice per group) were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. One week after second vaccination, splenocytes of mice were collected and assessed for E7-specific CD8+ T cells. (E-F) Mice (5 mice per group) were challenged with TC-1 cells. Three days after tumor challenge, mice were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. (E) Tumor growth was measured using digital caliper. (F) Mice survival were assessed using Kaplan-Meier analysis. Data are presented as mean ± SD (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

    Journal: Oncoimmunology

    Article Title: A novel function of API5 (apoptosis inhibitor 5), TLR4-dependent activation of antigen presenting cells

    doi: 10.1080/2162402X.2018.1472187

    Figure Lengend Snippet: DC activating effect of API5 is TLR4 dependent. Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein or LPS (100ng/ml). (A) Supernatants were collected and levels of various cytokines were assessed using ELISA. (B) Expression of various maturation markers (CD40, CD80, CD86 and MHCI) by treated cells was analyzed using flow cytometry. (C) Wild type, TLR2 KO or TLR4 KO murine DCs were treated with API5 (5μg/ml) protein for 0, 10, 30, or 60 minutes, lysed, and assessed for the expression of various MAPk and IkB-α using western blot analysis. (D) Mice (5 mice per group) were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. One week after second vaccination, splenocytes of mice were collected and assessed for E7-specific CD8+ T cells. (E-F) Mice (5 mice per group) were challenged with TC-1 cells. Three days after tumor challenge, mice were vaccinated with wild type or TLR4 KO DCs treated with API5 and pulsed with E7 peptide twice at one week intervals. (E) Tumor growth was measured using digital caliper. (F) Mice survival were assessed using Kaplan-Meier analysis. Data are presented as mean ± SD (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001).

    Article Snippet: Anti-human TLR4 antibody (Santa Cruz), recombinant TLR4 protein 0.5μg/μl (Sino Biological), recombinant API5 protein (0.075μg/μl or 0.0375μg/μl), or BSA (0.075μg/μl) were gradually incubated with protein A biosensor.

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Cytometry, Western Blot, Mouse Assay

    API5 as a DAMP that interacts with TLR4. (A) API5 expression level of various cancer cells were detected using western blot analysis. (B) To confirm that API5 protein is a DAMP, expression of API5 by cancer cells with or without doxorubicin treatment was measured using western blot analysis. (C) The production of recombinant human API5 protein via bacterial protein purification system was confirmed using CBB (coomassie brilliant blue) and western blot analysis. (D) Binding between API5 and TLR2 or TLR4 was assessed using immunoprecipitation. (E) TLR4 expressing HEK293 cells were transfected with plasmid encoding luciferase under NF-kB promoter, and treated with API5 (0.5μg/ml), GFP (0.5μg/ml) or LPS (100ng/ml), and assessed for luciferase signal. Data are presented as mean ± SD (** = p

    Journal: Oncoimmunology

    Article Title: A novel function of API5 (apoptosis inhibitor 5), TLR4-dependent activation of antigen presenting cells

    doi: 10.1080/2162402X.2018.1472187

    Figure Lengend Snippet: API5 as a DAMP that interacts with TLR4. (A) API5 expression level of various cancer cells were detected using western blot analysis. (B) To confirm that API5 protein is a DAMP, expression of API5 by cancer cells with or without doxorubicin treatment was measured using western blot analysis. (C) The production of recombinant human API5 protein via bacterial protein purification system was confirmed using CBB (coomassie brilliant blue) and western blot analysis. (D) Binding between API5 and TLR2 or TLR4 was assessed using immunoprecipitation. (E) TLR4 expressing HEK293 cells were transfected with plasmid encoding luciferase under NF-kB promoter, and treated with API5 (0.5μg/ml), GFP (0.5μg/ml) or LPS (100ng/ml), and assessed for luciferase signal. Data are presented as mean ± SD (** = p

    Article Snippet: Anti-human TLR4 antibody (Santa Cruz), recombinant TLR4 protein 0.5μg/μl (Sino Biological), recombinant API5 protein (0.075μg/μl or 0.0375μg/μl), or BSA (0.075μg/μl) were gradually incubated with protein A biosensor.

    Techniques: Expressing, Western Blot, Recombinant, Protein Purification, Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Luciferase

    CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells. ( a ) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. ( b ) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.

    Journal: Scientific Reports

    Article Title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4

    doi: 10.1038/srep24346

    Figure Lengend Snippet: CnB triggered cytokine secretion via the TLR4 signalling pathway in RAW264.7 macrophages, but not in SK-HEP-1 cells. ( a ) CnB induced the secretion of pro-inflammatory cytokines secretion through the MyD88-dependent pathway in the macrophage cell line. ( b ) CnB induced CCL5 and IFNβ secretion through the TRIF-dependent pathway in the macrophage cell line. The RAW264.7 cells were treated with 5, 25, and 100 μg/ml CnB for 24 h, and the SK-HEP-1 cells were treated with 200, 400, and 800 μg/ml CnB for 48 h. The amounts of secreted cytokines were determined by ELISA. Data represent mean ± s.e.m. from three independent experiments.

    Article Snippet: The recombinant extracellular domain of TLR4 (10146-H08B) and soluble CD14 10073-H007H) were purchased from Sino Biological, Inc.

    Techniques: Enzyme-linked Immunosorbent Assay

    The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization. ( a ) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). ( b ) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). ( c ) Positive correlation between CnB uptake and TLR4 expression. 5 × 10 5 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 10 6 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). ( d ) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). ( e,f ) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis ( e ) or FI ( f ), (scale bar 50 μm, 20×). ( g ) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P

    Journal: Scientific Reports

    Article Title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4

    doi: 10.1038/srep24346

    Figure Lengend Snippet: The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization. ( a ) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). ( b ) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). ( c ) Positive correlation between CnB uptake and TLR4 expression. 5 × 10 5 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 10 6 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). ( d ) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). ( e,f ) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis ( e ) or FI ( f ), (scale bar 50 μm, 20×). ( g ) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P

    Article Snippet: The recombinant extracellular domain of TLR4 (10146-H08B) and soluble CD14 10073-H007H) were purchased from Sino Biological, Inc.

    Techniques: Transfection, Incubation, Laser-Scanning Microscopy, Labeling, Fluorescence, Microscopy, Expressing, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot

    Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion. ( a ) The uptake of CnB could be inhibited by LPS. ( b ) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). ( c ) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. ( d ) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. ( e ) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 10 5 /well) and co-transfected with the pNF-κB-luc and pRL-null-Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P

    Journal: Scientific Reports

    Article Title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4

    doi: 10.1038/srep24346

    Figure Lengend Snippet: Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion. ( a ) The uptake of CnB could be inhibited by LPS. ( b ) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). ( c ) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. ( d ) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. ( e ) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 10 5 /well) and co-transfected with the pNF-κB-luc and pRL-null-Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P

    Article Snippet: The recombinant extracellular domain of TLR4 (10146-H08B) and soluble CD14 10073-H007H) were purchased from Sino Biological, Inc.

    Techniques: Binding Assay, Incubation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Labeling, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    MST measurements of the interaction between CnB and the TLR4 receptor complexes. ( a ) Measurement of rhCnB binding to the purified TLR4 ectodomain. ( b ) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. ( c ) Measurement of rhCnB binding to purified soluble CD14. ( d ) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. ( e ) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. ( f ) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. ( g ) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.

    Journal: Scientific Reports

    Article Title: Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4

    doi: 10.1038/srep24346

    Figure Lengend Snippet: MST measurements of the interaction between CnB and the TLR4 receptor complexes. ( a ) Measurement of rhCnB binding to the purified TLR4 ectodomain. ( b ) Measurement of CnB binding to the full-length TLR4 from transfected HEK293 lysates. ( c ) Measurement of rhCnB binding to purified soluble CD14. ( d ) Measurement of rhCnB binding to full-length membrane-anchored CD14 from transfected HEK293 lysates. ( e ) Measurement of the binding of rhCnB to secreted MD2 in the supernatant from transfected HEK293 cells. ( f ) Measurement of rhCnB binding to MD2 in the transfected Hek293 cell lysates. The purified TLR4 ectodomain or soluble CD14 was labelled with DyLight 488, and the concentration of labelled protein was adjusted to 20 nM. GFP-tagged TLR4, CD14 and MD2 constructs were transfected into HEK293 cells, incubated for 48 h, and lysed with RIPA buffer. Secreted MD2 was obtained from the supernatant of the MD2-transfected hek293 cells. The lysates were diluted according to fluorescence intensity. The recombinant CnB protein was dissolved to a 500 μM concentration using MST buffer and 16 1:1 dilution samples were prepared. The labelled proteins or GFP-tagged receptors lysates were added into each ligand dilution and mixed. After 10 min incubation, each solution was added to Standard Treated Capillaries for Thermophoresis. The data were analysed using NT. Analysis software. All data are representative of at least two independent experiments. ( g ) Model depicting the recognition of exogenous CnB. Membrane-anchored or soluble CD14 first recognized CnB and transported it to the TLR4/MD2 complex located on the plasma membrane, followed by internalization of the CnB/TLR4 receptor complexes and signalling through TLR4. Free MD2 also recognized and bound to CnB, although the affinity was lower than that of the MD2 on the plasma membrane.

    Article Snippet: The recombinant extracellular domain of TLR4 (10146-H08B) and soluble CD14 10073-H007H) were purchased from Sino Biological, Inc.

    Techniques: Binding Assay, Purification, Transfection, Concentration Assay, Construct, Incubation, Fluorescence, Recombinant, Software

    DENV NS1 binds to platelets and induces activation through TLR4 signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P

    Journal: PLoS Pathogens

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

    doi: 10.1371/journal.ppat.1007625

    Figure Lengend Snippet: DENV NS1 binds to platelets and induces activation through TLR4 signal transduction. (A) The protein expression levels of TLR4 and β actin, an internal control, in human-isolated platelets from 3 different donors were detected using Western blotting (50 μg protein/lane). (B) Human-isolated platelets were preincubated with αTLR4 or a control Rabbit IgG for 1 h, and the binding of NS1 on platelet surfaces was determined by flow cytometry using FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) (n = 3). (C) The binding of NS1 on platelets isolated from wild-type or TLR4 knockout mice was determined by flow cytometry (n = 4). Platelets were preincubated with or without different concentrations of (D) αTLR4 (E) the TLR4 antagonist LPS-Rs, (F) the TLR4 signaling inhibitor TAK242, or the LPS inhibitor polymyxin B (10 μg/ml) for 30 min, followed by BSA or NS1 (10 μg/ml) stimulation for 1 h (n = 4). The percent fluorescence of NS1 binding and the P-selectin surface expression on platelets were analyzed by FACSCalibur flow cytometry. *P

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) To examine the direct interaction between DENV NS1 and candidate receptors, 50 μl of TLR4 (purified from baculovirus-system, Sino Biological), TLR2 (purified from baculovirus-system, Sino Biological), His-taq protein or BSA (5 μg/ml) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4 °C overnight.

    Techniques: Activation Assay, Transduction, Expressing, Isolation, Western Blot, Binding Assay, Flow Cytometry, Knock-Out, Mouse Assay, Fluorescence

    TLR4 is involved in DENV-induced prolongation of bleeding time and hemorrhage in mice. A hemorrhagic mouse model was performed with C57BL/6J mice (WT) and TLR4 -/- C57BL/6J background mice as described in the Methods. (A) Mouse skin samples were removed to observe local hemorrhage on day 3 after DENV injection. The number of mice with hemorrhage divided by the total number of mice inoculated in each group is indicated. Yellow arrows indicate local skin hemorrhage. (B) The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity. (C) The tail bleeding time and (D) platelet counts were also determined on day 3 before sacrifice. *P

    Journal: PLoS Pathogens

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

    doi: 10.1371/journal.ppat.1007625

    Figure Lengend Snippet: TLR4 is involved in DENV-induced prolongation of bleeding time and hemorrhage in mice. A hemorrhagic mouse model was performed with C57BL/6J mice (WT) and TLR4 -/- C57BL/6J background mice as described in the Methods. (A) Mouse skin samples were removed to observe local hemorrhage on day 3 after DENV injection. The number of mice with hemorrhage divided by the total number of mice inoculated in each group is indicated. Yellow arrows indicate local skin hemorrhage. (B) The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity. (C) The tail bleeding time and (D) platelet counts were also determined on day 3 before sacrifice. *P

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) To examine the direct interaction between DENV NS1 and candidate receptors, 50 μl of TLR4 (purified from baculovirus-system, Sino Biological), TLR2 (purified from baculovirus-system, Sino Biological), His-taq protein or BSA (5 μg/ml) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4 °C overnight.

    Techniques: Mouse Assay, Injection

    Proposed mechanisms of the contribution of DENV NS1 to cause thrombocytopenia and hemorrhage during DENV infection. Circulating DENV NS1 binds to platelets via TLR4 or other molecules to induce the release of ADP, which in turn elevates P-selectin expression and PS exposure on platelet surfaces leading to platelet activation and enhancement of the platelet aggregation. In addition, NS1-activated platelets are prone to adhere onto endothelium or phagocytosis by macrophages. On the other hand, NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release. All these effects induced by NS1 can contribute to the thrombocytopenia and hemorrhage during DENV infection.

    Journal: PLoS Pathogens

    Article Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage

    doi: 10.1371/journal.ppat.1007625

    Figure Lengend Snippet: Proposed mechanisms of the contribution of DENV NS1 to cause thrombocytopenia and hemorrhage during DENV infection. Circulating DENV NS1 binds to platelets via TLR4 or other molecules to induce the release of ADP, which in turn elevates P-selectin expression and PS exposure on platelet surfaces leading to platelet activation and enhancement of the platelet aggregation. In addition, NS1-activated platelets are prone to adhere onto endothelium or phagocytosis by macrophages. On the other hand, NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release. All these effects induced by NS1 can contribute to the thrombocytopenia and hemorrhage during DENV infection.

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) To examine the direct interaction between DENV NS1 and candidate receptors, 50 μl of TLR4 (purified from baculovirus-system, Sino Biological), TLR2 (purified from baculovirus-system, Sino Biological), His-taq protein or BSA (5 μg/ml) in PBS (pH 7.3) was coated onto 96-well ELISA plates at 4 °C overnight.

    Techniques: Infection, Expressing, Activation Assay

    Effects of Danshensu on the protein expression of p-NF-κB p65 and TLR4 in SARS-CoV-2 S-induced lung tissues and the mRNA expression of ACE2 and AGT. a Western blotting of proteins involved in TLR4, NF-κB p65 and p-NF-κB p65. b The mRNA level of ACE2. c The mRNA level of AGT. Compared to SARS-CoV-2 S group, * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Danshensu alleviates pseudo-typed SARS-CoV-2 induced mouse acute lung inflammation

    doi: 10.1038/s41401-021-00714-4

    Figure Lengend Snippet: Effects of Danshensu on the protein expression of p-NF-κB p65 and TLR4 in SARS-CoV-2 S-induced lung tissues and the mRNA expression of ACE2 and AGT. a Western blotting of proteins involved in TLR4, NF-κB p65 and p-NF-κB p65. b The mRNA level of ACE2. c The mRNA level of AGT. Compared to SARS-CoV-2 S group, * P

    Article Snippet: Protein was extracted from cells and mouse lung tissues, SARS-CoV-2 Spike and SARS-CoV-2 Spike 2 (40590-T62 and 40589-V08B1, Sino Biological), TLR4 (BA1717, Boster Biological Technology, Wuhan, China), NF-κB p65 (8242 S, Cell Signaling Technology, Danvers, MA, USA), Phospho-NF-κB p65 (p-NF-κB p65) (3033, Cell Signaling Technology), and GAPDH (FD0063, Hangzhou Fude Biological Technology, Hangzhou, China), were added to membranes.

    Techniques: Expressing, Western Blot