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

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

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

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

Figure Lengend 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.

Article Snippet: 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.

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

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

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

Article Snippet: 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.

Techniques:

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

Figure Lengend 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.

Article Snippet: 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.

Techniques: Suspension, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

Figure Lengend 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.

Article Snippet: 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.

Techniques: Control, Incubation

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

Figure Lengend 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.

Article Snippet: 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.

Techniques: Isolation

Journal: Nature Communications

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

doi: 10.1038/s41467-024-53770-9

Figure Lengend 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.

Article Snippet: 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.

Techniques: Immunofluorescence, Microscopy, Transfection, Construct

Journal: Journal of periodontal research

Article Title: Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

doi: 10.1111/jre.12173

Figure Lengend Snippet: Fig. 1. Toll-like receptor 4 (TLR4) protein expression profiles in healthy gingiva (diam- inobenzidine stain; scale bars represent 200, 50 or 25 lm, as indicated). (a) Micrograph of healthy gingiva biopsy section: (b-e) show magnified regions. TLR4 immunoreactivity was found in the oral epithelium (OE) (b) and oral sulcular epithelium (OSE) (b, c, e), and occasionally in the junctional epithelium (JE) (d). Fibroblast-like cells (open arrows) (c, d) show slight TLR4 immunoreactivity. Leukocyte-like cells (open and filled arrowheads) were scattered in the OSE, JE and subepithelial regions of the healthy gingiva (c, d). Most leukocyte-like cells, including those infiltrating the epithelium, were not TLR4 immunore- active (open arrowheads), except for those with dendritic morphology, which were TLR4 positive (fillled arrowheads). The TLR4-positive staining in endothelial cells (arrows) was very weak or absent.

Article Snippet: After blocking in 2.5% horse serum for 20 min, the sections were incubated with primary antibodies against TLR4 (rabbit antihuman polyclonal IgG, 1 : 100 dilution, sc-10741; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 1% bovine albumin at 4°C overnight, then stained (ImmPRESSTM Universal Reagent, MP-7500; Vector Laboratories, Burlingame, CA, USA) and visualized using a diaminobenzidine solution.

Techniques: Expressing, Staining

Journal: Journal of periodontal research

Article Title: Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

doi: 10.1111/jre.12173

Figure Lengend Snippet: Fig. 2. Toll-like receptor 4 (TLR4) protein-expression profiles in shallow (A) or deep (B) periodontal pockets (diaminobenzidine stain; scale bars represent 200, 50 or 25 lm, as indi- cated). (a) Section of shallow periodontal pocket biopsy: (b–d) show magnified regions. (e) Section of deep periodontal pocket biopsy: (f–k) show magnified regions. TLR4 immuno- reactivity could be found in oral epithelium (OE) (d) and pocket epithelium (PE) (c, g). Com- pared with healthy gingiva (Fig. 1c and 1d), where fibroblasts were mostly free of TLR4 immunoreactivity, fibroblast-like cells in periodontitis tissue (f, i; open arrows) proximal to inflammatory front were heavily stained. Leukocyte-like cells (open and filled arrowhead) appeared to densely infiltrate the periodontitis tissue (f, j). Although most epithelium-infil- trating leukocyte-like cells were not TLR4 immunoreactive (open arrowhead), those in the subepithelial region were more strongly stained (solid arrowhead) than in healthy gingiva (Fig. 1d). TLR4-positive inflammatory cells appeared to accumulate around the front of the periodontitis lesion (h, k), which was characterized by a mass of densely infiltrating leuko- cytes. The proportion of TLR4-positive subepithelial cells in periodontitis tissue was signifi- cantly greater than that in healthy controls (compared with Fig. 1: 68.4 13.7% in 38 periodontal pockets vs. 12.3 5.6% in 10 control samples; p < 0.001, t-test.).

Article Snippet: After blocking in 2.5% horse serum for 20 min, the sections were incubated with primary antibodies against TLR4 (rabbit antihuman polyclonal IgG, 1 : 100 dilution, sc-10741; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 1% bovine albumin at 4°C overnight, then stained (ImmPRESSTM Universal Reagent, MP-7500; Vector Laboratories, Burlingame, CA, USA) and visualized using a diaminobenzidine solution.

Techniques: Expressing, Staining, Control

Journal: Journal of periodontal research

Article Title: Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

doi: 10.1111/jre.12173

Figure Lengend Snippet: Fig. 4. Expression of Toll-like receptor 4 (TLR4), lymphocyte antigen 96 (MD-2), cluster of differentiation 14 (CD14) and lipopolysaccharide-binding protein (LBP) mRNAs in pri- mary human gingival keratinocytes (HGK), human gingival fibroblasts (HGF) and healthy gingival tissue (HGT). (A) The human TLR4 gene includes two exons, a and b, which yield four possible splicing variants. (B) Possible splicing variants TLR4.1, TLR4.2, TLR4.3 and TLR4.4. A pair of PCR primers (P1 and P2) was designed to amplify the splicing variants containing exons a and b. (C) Three TLR4 splicing variants, TLR4.3, TLR4.1 and TLR4.4, were found in HGF, HGK and HGT. (D) Two mRNA splicing vari- ants of MD-2 – MD-2.1 (containing the splicing exon) and MD-2.2 (lacking the splicing exon) – were found in HGF, HGK and HGT. MD-2.1 appears to be expressed more in gingival cells and tissue. (E) CD14 mRNA was detected in HGF, HGK and HGT. (F) LBP mRNA was found in HGT, but not in HGK or HGF.

Article Snippet: After blocking in 2.5% horse serum for 20 min, the sections were incubated with primary antibodies against TLR4 (rabbit antihuman polyclonal IgG, 1 : 100 dilution, sc-10741; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 1% bovine albumin at 4°C overnight, then stained (ImmPRESSTM Universal Reagent, MP-7500; Vector Laboratories, Burlingame, CA, USA) and visualized using a diaminobenzidine solution.

Techniques: Expressing, Binding Assay

Journal: Journal of periodontal research

Article Title: Differential expression of Toll-like receptor 4 in healthy and diseased human gingiva.

doi: 10.1111/jre.12173

Figure Lengend Snippet: Fig. 5. Expression of cluster of differentiation 14 (CD14), lymphocyte antigen 96 (MD-2) and Toll-like receptor 4 (TLR4) proteins in human gingival tissue and cells (A) and associ- ation between CD14, MD-2 and TLR4 peptides expressed in human gingival fibroblasts (HGF) and human gingival keratinocytes (HGK) (B). Proteins were immunoprecipitated (IP) and immunoblotted (IB) from the whole cell or tissue lysate (500 lg of cellular pro- tein or 800 lg of tissue protein for TLR4 and CD14, and 20 lg of protein for MD-2). The anti-human antibody species were rabbit, mouse and goat. (A) Expression of (a) CD14, (b) MD-2 and (c) TLR4 protein in human peripheral blood mononuclear cells (PBMC) (positive control), HGF, HGK, healthy gingival tissue (HGT) and periodontitis gingival tissue (PGT). BSA: bovine serum albumin (500 lg) negative-control lane. (B) Association between CD14, MD-2 and TLR4 proteins expressed in HGF and HGK. Whole HGF, HGK and PBMC (positive control) lysates (each containing 500 lg of pro- tein) and BSA (500 lg; negative control) were IP with anti-CD14 (e), -MD-2 (g) or -TLR4 (d, f) antibody, and IB with the relevant anti-human antibody. (d) and (e) TLR4 and CD14 peptides were co-immunoprecipitated. (f) and (g) MD-2 and TLR4 peptides were co-immunoprecipitated. CD14 and MD-2 did not co-immunoprecipitate (data not shown), indicating that these molecules do not directly interact with each other. Gt, goat; Ms, mouse; Rb, rabbit.

Article Snippet: After blocking in 2.5% horse serum for 20 min, the sections were incubated with primary antibodies against TLR4 (rabbit antihuman polyclonal IgG, 1 : 100 dilution, sc-10741; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) in 1% bovine albumin at 4°C overnight, then stained (ImmPRESSTM Universal Reagent, MP-7500; Vector Laboratories, Burlingame, CA, USA) and visualized using a diaminobenzidine solution.

Techniques: Expressing, Immunoprecipitation, Positive Control, Negative Control

Journal: Experimental & Molecular Medicine

Article Title: Interrupting oral infection of Porphyromonas gingivalis with anti-FimA antibody attenuates bacterial dissemination to the arthritic joint and improves experimental arthritis

doi: 10.1038/emm.2017.301

Figure Lengend Snippet: Porphyromonas gingivalis (Pg) is involved in synovial inflammation. ( a , b ) Double immunofluorescence staining of Toll like receptor-2 (TLR2) ( a ; red) or TLR4 ( b ; red) and FimA (green) in the joint of collagen-induced arthritis (CIA) mice. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 μm. ( c , d ) Rheumatoid arthritis synovial fibroblast (RASF) was treated with Pg ( c ) or crude fimbriae ( d ) and incubated for various time points. The RNA expression levels of synovial inflammation-related cytokines and proteins were detected by real-time PCR. Statistical analysis was performed using one-way analysis of variance (ANOVA; n =3, * P <0.05 and ** P <0.01).

Article Snippet: Toll like receptor-2 (TLR2) and TLR4 were stained with anti-TLR2 Ab (Santa Cruz) and anti-TLR4 Ab (Santa Cruz), respectively, followed by Fluorescein rabbit anti-goat Ab (Vector Laboratories) in the joint sections.

Techniques: Double Immunofluorescence Staining, Staining, Incubation, RNA Expression, Real-time Polymerase Chain Reaction

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

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: