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human tlr4  (Addgene inc)


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    Structured Review

    Addgene inc human tlr4
    a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of <t>TLR4</t> signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.
    Human Tlr4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/tlr4+expression+vector/hTLR4+(Plasmid+%2313086)/pmc11558003-357-12-20
    Average 93 stars, based on 18 article reviews
    human tlr4 - by Bioz Stars, 2026-11
    93/100 stars

    Images

    1) Product Images from "Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling"

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

    Journal: Nature Communications

    doi: 10.1038/s41467-024-53770-9

    a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.
    Figure Legend Snippet: a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.

    Techniques Used: Cell Culture, Refractive Index, IF-cells, Concentration Assay, Control, Incubation, Generated

    Pharmacological parameter of LPS induced DMR at six selected time points in HEK293  TLR4/MD-2/CD14  reporter cells
    Figure Legend Snippet: Pharmacological parameter of LPS induced DMR at six selected time points in HEK293 TLR4/MD-2/CD14 reporter cells

    Techniques Used:

    a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

    Techniques Used: Suspension, Two Tailed Test

    THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Control, Incubation

    a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

    Techniques Used: Isolation

    a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.
    Figure Legend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

    Techniques Used: Immunofluorescence, Microscopy, Transfection, Construct

    Related Articles

    Transfection:

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling
    Article Snippet: Cells were transfected with the plasmid by using PEI (Polysciences) and were cultured at 37 °C in a humidified atmosphere with 5% CO 2 . .. Next, cells were transiently transfected with or without 50 ng of the human TLR4 (hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid # 13086; http://n2t.net/addgene:13086 ; RRID:Addgene_13086), 10 ng of CD14 (pcDNA3.1-hDC14, a gift from Douglas Golenbock, Addgene plasmid # 13645; http://n2t.net/addgene:13645 ; RRID:Addgene_13645), 10 ng of MD-2 expression vector (pFlag-CMV1-hMD2, a gift from Douglas Golenbock, Addgene plasmid # 13028; http://n2t.net/addgene:13028 ; RRID:Addgene_13028), 15 ng of the endothelial leukocyte adhesion molecule (ELAM) firefly luciferase reporter vector (pGL3-ELAM-luc, a gift from Douglas Golenbock, Addgene plasmid # 13029; http://n2t.net/addgene:13029 ; RRID:Addgene_13029), 15 ng Renilla luciferase control vector or empty pcDNA3.1 vector. ..

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils.
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Plasmid Preparation:

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling
    Article Snippet: Cells were transfected with the plasmid by using PEI (Polysciences) and were cultured at 37 °C in a humidified atmosphere with 5% CO 2 . .. Next, cells were transiently transfected with or without 50 ng of the human TLR4 (hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid # 13086; http://n2t.net/addgene:13086 ; RRID:Addgene_13086), 10 ng of CD14 (pcDNA3.1-hDC14, a gift from Douglas Golenbock, Addgene plasmid # 13645; http://n2t.net/addgene:13645 ; RRID:Addgene_13645), 10 ng of MD-2 expression vector (pFlag-CMV1-hMD2, a gift from Douglas Golenbock, Addgene plasmid # 13028; http://n2t.net/addgene:13028 ; RRID:Addgene_13028), 15 ng of the endothelial leukocyte adhesion molecule (ELAM) firefly luciferase reporter vector (pGL3-ELAM-luc, a gift from Douglas Golenbock, Addgene plasmid # 13029; http://n2t.net/addgene:13029 ; RRID:Addgene_13029), 15 ng Renilla luciferase control vector or empty pcDNA3.1 vector. ..

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils.
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Article Title: Haptoglobin buffers lipopolysaccharides to delay activation of NFκB
    Article Snippet: Amicon filters were from Merck-Millipore. .. The hTLR4 expression vector (Addgene 13086) was a gift from Ruslan Medzhitov. .. pcDNA3-CD14 (Addgene 13645) and pFlag-CMV1-hMD2 (Addgene 13028) were gifts from Doug Golenbock.

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Expressing:

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling
    Article Snippet: Cells were transfected with the plasmid by using PEI (Polysciences) and were cultured at 37 °C in a humidified atmosphere with 5% CO 2 . .. Next, cells were transiently transfected with or without 50 ng of the human TLR4 (hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid # 13086; http://n2t.net/addgene:13086 ; RRID:Addgene_13086), 10 ng of CD14 (pcDNA3.1-hDC14, a gift from Douglas Golenbock, Addgene plasmid # 13645; http://n2t.net/addgene:13645 ; RRID:Addgene_13645), 10 ng of MD-2 expression vector (pFlag-CMV1-hMD2, a gift from Douglas Golenbock, Addgene plasmid # 13028; http://n2t.net/addgene:13028 ; RRID:Addgene_13028), 15 ng of the endothelial leukocyte adhesion molecule (ELAM) firefly luciferase reporter vector (pGL3-ELAM-luc, a gift from Douglas Golenbock, Addgene plasmid # 13029; http://n2t.net/addgene:13029 ; RRID:Addgene_13029), 15 ng Renilla luciferase control vector or empty pcDNA3.1 vector. ..

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils.
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Article Title: Haptoglobin buffers lipopolysaccharides to delay activation of NFκB
    Article Snippet: Amicon filters were from Merck-Millipore. .. The hTLR4 expression vector (Addgene 13086) was a gift from Ruslan Medzhitov. .. pcDNA3-CD14 (Addgene 13645) and pFlag-CMV1-hMD2 (Addgene 13028) were gifts from Doug Golenbock.

    Article Title: Extracellular vimentin is a damage-associated molecular pattern protein serving as an agonist of TLR4 in human neutrophils
    Article Snippet: .. 20 h after plating, cells were transfected with plasmid DNA encoding for expression of miRFP720 (gift from Leonard Lab, pPD1039), hTLR4 (Addgene #13086), hMD2 (Geneblock in pcDNA, see below), and pcDNA, totaling 600 ng DNA/well using 0.75 uL/well Lipofectamine 3000. ..

    Luciferase:

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling
    Article Snippet: Cells were transfected with the plasmid by using PEI (Polysciences) and were cultured at 37 °C in a humidified atmosphere with 5% CO 2 . .. Next, cells were transiently transfected with or without 50 ng of the human TLR4 (hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid # 13086; http://n2t.net/addgene:13086 ; RRID:Addgene_13086), 10 ng of CD14 (pcDNA3.1-hDC14, a gift from Douglas Golenbock, Addgene plasmid # 13645; http://n2t.net/addgene:13645 ; RRID:Addgene_13645), 10 ng of MD-2 expression vector (pFlag-CMV1-hMD2, a gift from Douglas Golenbock, Addgene plasmid # 13028; http://n2t.net/addgene:13028 ; RRID:Addgene_13028), 15 ng of the endothelial leukocyte adhesion molecule (ELAM) firefly luciferase reporter vector (pGL3-ELAM-luc, a gift from Douglas Golenbock, Addgene plasmid # 13029; http://n2t.net/addgene:13029 ; RRID:Addgene_13029), 15 ng Renilla luciferase control vector or empty pcDNA3.1 vector. ..

    Control:

    Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling
    Article Snippet: Cells were transfected with the plasmid by using PEI (Polysciences) and were cultured at 37 °C in a humidified atmosphere with 5% CO 2 . .. Next, cells were transiently transfected with or without 50 ng of the human TLR4 (hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid # 13086; http://n2t.net/addgene:13086 ; RRID:Addgene_13086), 10 ng of CD14 (pcDNA3.1-hDC14, a gift from Douglas Golenbock, Addgene plasmid # 13645; http://n2t.net/addgene:13645 ; RRID:Addgene_13645), 10 ng of MD-2 expression vector (pFlag-CMV1-hMD2, a gift from Douglas Golenbock, Addgene plasmid # 13028; http://n2t.net/addgene:13028 ; RRID:Addgene_13028), 15 ng of the endothelial leukocyte adhesion molecule (ELAM) firefly luciferase reporter vector (pGL3-ELAM-luc, a gift from Douglas Golenbock, Addgene plasmid # 13029; http://n2t.net/addgene:13029 ; RRID:Addgene_13029), 15 ng Renilla luciferase control vector or empty pcDNA3.1 vector. ..



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    Addgene inc expression vectors for tlr4
    A) Schematic representation of LPS transfer from Cd14 to the <t>Tlr4/Md-2</t> complex. LPS (yellow) is brought by Cd14 (purple) and loaded into Md-2 (navy blue). Md-2 is bound by Tlr4 (cyan). Binding of LPS to the Md-2 co-receptor causes dimerization of the Tlr4/Md-2 complex, activating a downstream inflammatory response. B) The interface between <t>human</t> <t>Tlr4</t> (cyan) and Md-2 (navy blue) is extensive. Both are required to form a productive interaction with LPS (yellow). Structure shown was made from PDB 3FXI .
    Expression Vectors For Tlr4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc tlr4 expression vectors
    Fig. 5. oxLDL induces uPAR association with <t>TLR4.</t> A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).
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    Eisai Inc hek 293 cells stably transfected with both tlr4 and md2 expression vectors
    Fig. 5. oxLDL induces uPAR association with <t>TLR4.</t> A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).
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    Tularik Inc expression vectors for the nh2 terminus of human flag-tagged tlr4
    Fig. 5. oxLDL induces uPAR association with <t>TLR4.</t> A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).
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    Image Search Results


    NF-κB Reporter Assays with TLR4, MD-2, and CD14. Human embryonic kidney 293 (HEK293) cells were transfected with plasmid expression vectors for Firefly NF-κB and Renilla luciferase reporters, plus CD14, MD-2, and TLR4 (gray bar); MD-2 and TLR4 (no CD14, orange bar); CD14 and TLR4 (no MD-2, blue bar); or CD14 and MD-2 (no TLR4, green bar). Twenty-four hours after transfection, cells were incubated with media (Control), 10 μM heme, 10 ng/ml LPS, or heme + LPS for 6 h in media containing 1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Results are representative of 3 independent experiments run in triplicate. Bars are means + SD. # P < 0.01 control vs. heme, LPS, and heme + LPS. * P < 0.01 TLR4 + MD-2 + CD14 (gray bar) vs. no CD14 (purple bar), no MD-2 (orange bar), and no TLR4 (green bar) for each stimulant, using One Way ANOVA with Holm-Sidac correction.

    Journal: Frontiers in Immunology

    Article Title: Identification of a Heme Activation Site on the MD-2/TLR4 Complex

    doi: 10.3389/fimmu.2020.01370

    Figure Lengend Snippet: NF-κB Reporter Assays with TLR4, MD-2, and CD14. Human embryonic kidney 293 (HEK293) cells were transfected with plasmid expression vectors for Firefly NF-κB and Renilla luciferase reporters, plus CD14, MD-2, and TLR4 (gray bar); MD-2 and TLR4 (no CD14, orange bar); CD14 and TLR4 (no MD-2, blue bar); or CD14 and MD-2 (no TLR4, green bar). Twenty-four hours after transfection, cells were incubated with media (Control), 10 μM heme, 10 ng/ml LPS, or heme + LPS for 6 h in media containing 1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Results are representative of 3 independent experiments run in triplicate. Bars are means + SD. # P < 0.01 control vs. heme, LPS, and heme + LPS. * P < 0.01 TLR4 + MD-2 + CD14 (gray bar) vs. no CD14 (purple bar), no MD-2 (orange bar), and no TLR4 (green bar) for each stimulant, using One Way ANOVA with Holm-Sidac correction.

    Article Snippet: In the morning, cells were transiently transfected with 50 ng TLR4 expression vector (pcDNA3.1-hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid #13086) or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector (pNifty-Luc, InvivoGen), 15 ng of Renilla luciferase control vector (pRL-TK, Promega), 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector (pCDNA3.1-hCD14, a gift from Doug Golenbock, Addgene plasmid #13645) per well using Lipofectamine Plus reagent (LifeTechnology).

    Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Control, Activity Assay

    NF-κB reporter assays with WT and mutant MD-2. HEK293 cells were transfected with plasmid expression vectors for WT or mutant MD-2, TLR4, CD14, Firefly luciferase NF-κB reporter, and Renilla luciferase control. Twenty-four hours after transfection, cells were treated with 10 μM heme or 10 ng/ml LPS for 6 h in media containing 0.1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Luciferase activity is expressed as a percent of cells transfected with WT MD-2 stimulated with heme or LPS (100% activity, black bars and horizontal dashed black line). Magenta cluster and green cluster refer to the 2 clusters of potential heme binding amino acids in . Results are representative of 3 independent experiments run in triplicate. Bar values are means ± SD. ** P < 0.01 vs. WT MD-2 using Kruskal-Wallis One Way Analysis of Variance on Ranks.

    Journal: Frontiers in Immunology

    Article Title: Identification of a Heme Activation Site on the MD-2/TLR4 Complex

    doi: 10.3389/fimmu.2020.01370

    Figure Lengend Snippet: NF-κB reporter assays with WT and mutant MD-2. HEK293 cells were transfected with plasmid expression vectors for WT or mutant MD-2, TLR4, CD14, Firefly luciferase NF-κB reporter, and Renilla luciferase control. Twenty-four hours after transfection, cells were treated with 10 μM heme or 10 ng/ml LPS for 6 h in media containing 0.1% serum. After 6 h, cells were lysed and luciferase activity was measured and expressed as the ratio of Firefly luciferase NF-κB reporter to Renilla luciferase control. Luciferase activity is expressed as a percent of cells transfected with WT MD-2 stimulated with heme or LPS (100% activity, black bars and horizontal dashed black line). Magenta cluster and green cluster refer to the 2 clusters of potential heme binding amino acids in . Results are representative of 3 independent experiments run in triplicate. Bar values are means ± SD. ** P < 0.01 vs. WT MD-2 using Kruskal-Wallis One Way Analysis of Variance on Ranks.

    Article Snippet: In the morning, cells were transiently transfected with 50 ng TLR4 expression vector (pcDNA3.1-hTLR4, a gift from Ruslan Medzhitov, Addgene plasmid #13086) or empty vector, along with 15 ng of Firefly luciferase NF-κB reporter vector (pNifty-Luc, InvivoGen), 15 ng of Renilla luciferase control vector (pRL-TK, Promega), 10 ng of wt or mutant MD-2 expression vector (pFlag-CMV1-hMD2), and 10 ng of CD14 expression vector (pCDNA3.1-hCD14, a gift from Doug Golenbock, Addgene plasmid #13645) per well using Lipofectamine Plus reagent (LifeTechnology).

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Luciferase, Control, Activity Assay, Binding Assay

    A) Schematic representation of LPS transfer from Cd14 to the Tlr4/Md-2 complex. LPS (yellow) is brought by Cd14 (purple) and loaded into Md-2 (navy blue). Md-2 is bound by Tlr4 (cyan). Binding of LPS to the Md-2 co-receptor causes dimerization of the Tlr4/Md-2 complex, activating a downstream inflammatory response. B) The interface between human Tlr4 (cyan) and Md-2 (navy blue) is extensive. Both are required to form a productive interaction with LPS (yellow). Structure shown was made from PDB 3FXI .

    Journal: bioRxiv

    Article Title: Identification and characterization of zebrafish Tlr4 co-receptor Md-2

    doi: 10.1101/817528

    Figure Lengend Snippet: A) Schematic representation of LPS transfer from Cd14 to the Tlr4/Md-2 complex. LPS (yellow) is brought by Cd14 (purple) and loaded into Md-2 (navy blue). Md-2 is bound by Tlr4 (cyan). Binding of LPS to the Md-2 co-receptor causes dimerization of the Tlr4/Md-2 complex, activating a downstream inflammatory response. B) The interface between human Tlr4 (cyan) and Md-2 (navy blue) is extensive. Both are required to form a productive interaction with LPS (yellow). Structure shown was made from PDB 3FXI .

    Article Snippet: Mammalian expression vectors containing human TLR4 and mouse Tlr4 were obtained from Addgene (#13085 and #13086), originally deposited by Ruslan Medzhitov.

    Techniques: Binding Assay

    A) Maximum likelihood phylogeny for 453 Tlr4 and Cd180 protein sequences. SH supports are indicated on the tree. Wedges are clades, with the length indicating the maximum branch length from the ancestor of the clade. The taxonomic distribution and number of genes within each wedge are indicated on the plot. B-G) Hits for human (purple) and zebrafish (orange) gene sets on six representative chromosomes taken from five species. The species and chromosome are indicated at the top of each plot. The x-axis denotes position on the chromosome. Triangles indicate gene start positions. The green arrow indicates the location of a Tlr4 gene. The y-axis is a running average of the BLAST e-value for each gene set along the genome (see methods). The numbers on the plot indicate the number of human and zebrafish hits within the region shown. H,I) Each row shows the chromosome with the most BLAST hits from the human (panel H) or zebrafish (panel I) gene set. Columns indicate specific genes from the set, with names denoted below. A colored square indicates a gene found somewhere on the chromosome. A green square is a tlr4 gene. The species tree is shown on the left; the chromosome number is on the right. J) Schematic representation of a plausible scenario for the history of the tlr4 gene. Times are taken from Hughes et al. and timetree.org .

    Journal: bioRxiv

    Article Title: Identification and characterization of zebrafish Tlr4 co-receptor Md-2

    doi: 10.1101/817528

    Figure Lengend Snippet: A) Maximum likelihood phylogeny for 453 Tlr4 and Cd180 protein sequences. SH supports are indicated on the tree. Wedges are clades, with the length indicating the maximum branch length from the ancestor of the clade. The taxonomic distribution and number of genes within each wedge are indicated on the plot. B-G) Hits for human (purple) and zebrafish (orange) gene sets on six representative chromosomes taken from five species. The species and chromosome are indicated at the top of each plot. The x-axis denotes position on the chromosome. Triangles indicate gene start positions. The green arrow indicates the location of a Tlr4 gene. The y-axis is a running average of the BLAST e-value for each gene set along the genome (see methods). The numbers on the plot indicate the number of human and zebrafish hits within the region shown. H,I) Each row shows the chromosome with the most BLAST hits from the human (panel H) or zebrafish (panel I) gene set. Columns indicate specific genes from the set, with names denoted below. A colored square indicates a gene found somewhere on the chromosome. A green square is a tlr4 gene. The species tree is shown on the left; the chromosome number is on the right. J) Schematic representation of a plausible scenario for the history of the tlr4 gene. Times are taken from Hughes et al. and timetree.org .

    Article Snippet: Mammalian expression vectors containing human TLR4 and mouse Tlr4 were obtained from Addgene (#13085 and #13086), originally deposited by Ruslan Medzhitov.

    Techniques:

    Fig. 5. oxLDL induces uPAR association with TLR4. A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).

    Journal: Journal of molecular and cellular cardiology

    Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

    doi: 10.1016/j.yjmcc.2013.11.005

    Figure Lengend Snippet: Fig. 5. oxLDL induces uPAR association with TLR4. A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).

    Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

    Techniques: Immunoprecipitation, Immunocytochemistry, Staining, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    Fig. 6. oxLDL-induced cytokine expression is mediated by NF-κB. A. VSMC were lentivirus-infected to express Gaussia luciferase under control of NF-κB responsive promoter as described in the Materials and methods section. 24 h after infection cells were transfected with SiCo or other siRNA as indicated. 24 h after transfection cells were stimulated with 5 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. B. HEK 293 cells were lentiviral infected to express Gaussia luciferase under control of NF-κB responsive promoter. Expression of uPAR was achieved by lentiviral infection. TLR4 and CD36 expression was achieved by cell transfection. Cells were stimulated with 8 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. C. Cells were stimulated with oxLDL in the presence of proteasome inhibitors. G-CSF and GM-CSF expression was assessed by RT-PCR. D. oxLDL- induced G-CSF and GM-CSF expression in the presence of NF-κB inhibitor BAY-11-7085 was assessed by RT-PCR. E. Primary human monocytes and macrophages were treated with conditioned medium of SiCo- and uPARsi-transfected VSMC stimulated with 8 μg/ml oxLDL. MCP-1 release was followed using Human CCL2/MCP-1 Quantikine ELISA Kit.* indicates significant difference (P b 0.05).

    Journal: Journal of molecular and cellular cardiology

    Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

    doi: 10.1016/j.yjmcc.2013.11.005

    Figure Lengend Snippet: Fig. 6. oxLDL-induced cytokine expression is mediated by NF-κB. A. VSMC were lentivirus-infected to express Gaussia luciferase under control of NF-κB responsive promoter as described in the Materials and methods section. 24 h after infection cells were transfected with SiCo or other siRNA as indicated. 24 h after transfection cells were stimulated with 5 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. B. HEK 293 cells were lentiviral infected to express Gaussia luciferase under control of NF-κB responsive promoter. Expression of uPAR was achieved by lentiviral infection. TLR4 and CD36 expression was achieved by cell transfection. Cells were stimulated with 8 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. C. Cells were stimulated with oxLDL in the presence of proteasome inhibitors. G-CSF and GM-CSF expression was assessed by RT-PCR. D. oxLDL- induced G-CSF and GM-CSF expression in the presence of NF-κB inhibitor BAY-11-7085 was assessed by RT-PCR. E. Primary human monocytes and macrophages were treated with conditioned medium of SiCo- and uPARsi-transfected VSMC stimulated with 8 μg/ml oxLDL. MCP-1 release was followed using Human CCL2/MCP-1 Quantikine ELISA Kit.* indicates significant difference (P b 0.05).

    Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

    Techniques: Expressing, Infection, Luciferase, Control, Transfection, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Fig. 7. In vivo evidence for uPAR-mediated events. A. Images captured from sub-fibrous cap area of the plaque stained for CD36 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. B. Images captured from sub-fibrous cap area of the plaque stained for TLR4 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. Scale bar 100 μm. * indicates significant difference (P b 0.05).

    Journal: Journal of molecular and cellular cardiology

    Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

    doi: 10.1016/j.yjmcc.2013.11.005

    Figure Lengend Snippet: Fig. 7. In vivo evidence for uPAR-mediated events. A. Images captured from sub-fibrous cap area of the plaque stained for CD36 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. B. Images captured from sub-fibrous cap area of the plaque stained for TLR4 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. Scale bar 100 μm. * indicates significant difference (P b 0.05).

    Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

    Techniques: In Vivo, Staining