Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Primary peritoneal macrophages from uPAR−/− mice were stimulated with suPAR with or without LPS for 15 min. Then, cells were fixed and stained for TLR4 (Alexa 488, green) and uPAR (Alexa 647, red). DAPI was used as nuclear stain. Scale bar 10 μm. B. Primary WT and uPAR−/− macrophages were stimulated with 100 ng/ml LPS and1μg/ml suPAR for 3 hrs C. Raw 264.7 cells were stimulated with 100 ng/ml LPS, fixed and stained as in A. Scale bar 12.5 μm. D. Raw 264.7 were stimulated with 1μg/ml biotin-LPS for 30 min, then cell lysis was performed. Protein complexes were precipitated using Streptavidin magnetic beads and analyzed by western blotting using anti-murine-uPAR antibody.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Staining, Lysis, Magnetic Beads, Western Blot

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Biotin-LPS binding was assessed in SiCo and uPARsi HK-2 cells as described. B. Duolink proximity ligation assay to assess uPAR/TLR4 and uPAR/CD36 interaction was performed on HK-2 cells stimulated with LPS for 15 min as described in Methods. C. Duolink images were quantified using Particles analysis tool of ImageJ. D. HK-2 cells were stimulated with LPS for 3 hrs after cell pre-treatment with CD36 inhibitor SSO. Expression of IL-6 and IL-8 was assessed by TaqMan RT-PCR.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Peritoneum of sham and LPS-injected WT mice was fixed and stained for uPAR and TLR4. DAPI used as nuclear stain­ scale bar 100μm. B. Expression of TNFα, MCP-1, IL-6 and IL-1 0 was assessed in mouse blood plasma before and 20 h after CLP surgery using Cytometric Beads Array. C: IL-6/IL-10 ratio in CLP mice 20 hrs after surgery.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Injection, Staining, Expressing, Clinical Proteomics

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: TLR4 mediates IL-8 expression in response to FnIII-1c and LPS in dermal fibroblasts. Monolayers of human dermal fibroblasts in 10% FBS/DMEM were treated for 24 h with ( A ) FnIII-1c or FnIII-13 (1-20 µg/mL), ( B ) LPS (1-100 ng/mL), ( C ) LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of the designated amounts of blocking antibody to TLR4 or TLR2. IgG served as control. ( D ) TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the TLR4 inhibitor, TAK-242. The wells without antibodies ( C ) or inhibitors ( D ) were set as 100%. IL-8 concentration in conditioned medium was determined by ELISA. The data represent the mean ± S.E. of triplicate assays from three separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Blocking Assay, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Role of TLR4 Receptor Complex in the Regulation of the Innate Immune Response by Fibronectin

doi: 10.3390/cells9010216

Figure Lengend Snippet: FnIII-1c-induced IL-8 expression requires membrane CD14. HEK cells expressing either TLR4/MD2 or TLR4/MD2/CD14 were incubated for 24 hours with the designated concentrations of LPS ( A , B ) or FnIII-1c ( C , D ) in either 10% FBS/DMEM ( A , C ) or 0.1% BSA/DMEM ( B , D ). ( E ) HEK-293 cells expressing TLR4-MD2 were treated with 1 µg/mL LPS or 20 µM FnIII-1c in 0.1% BSA/DMEM in the presence of the indicated concentration of exogenous soluble CD14 for 24 h. IL-8 concentration in the conditioned medium was measured by ELISA. The data represent the mean ± S.E. of triplicate assays from two ( A – D ) or three ( E ) separate experiments.

Article Snippet: Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Membrane, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Oxidized Low Density Lipoprotein Induces Bone Morphogenetic Protein-2 in Coronary Artery Endothelial Cells via Toll-like Receptors 2 and 4

doi: 10.1074/jbc.m110.214619

Figure Lengend Snippet: FIGURE 2. Neutralization of TLR2 or TLR4, but not LOX-1, reduces oxLDL-induced BMP-2 expression. A and B, representative immunoblots of three separateexperimentsanddensitometricdatashowthatneutralizationofTLR2orTLR4markedlyreducedBMP-2levelsafteroxLDLstimulation(80g/ml,24h). C and D, representative immunoblots of three separate experiments and densitometric data show that PGN (10 g/ml, 24 h) and LPS (200 ng/ml, 24 h) also induced BMP-2 expression and that their effects were greatly reduced by neutralizing antibodies against the receptors. E, representative immunoblot of three separate experiments and densitometric data show that neutralization of LOX-1 had no influence on oxLDL-induced BMP-2 expression (80 g/ml oxLDL, 24 h). Densitometry data are expressed as mean S.E. (error bars). n 3; *, p 0.05 versus control; #, p 0.05 versus correspondent treatment without receptor neutralization.

Article Snippet: Monoclonal neutralizing antibodies to human TLR2 and TLR4 were purchased from Imgenex (San Diego, CA).

Techniques: Neutralization, Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Oxidized Low Density Lipoprotein Induces Bone Morphogenetic Protein-2 in Coronary Artery Endothelial Cells via Toll-like Receptors 2 and 4

doi: 10.1074/jbc.m110.214619

Figure Lengend Snippet: FIGURE 3. Silencing of TLR2 or TLR4 reduces BMP-2 expression induced by oxLDL. A and B, representative immunoblots and densitometric data show that treatmentwithspecificsiRNAfor48hreducedcellularTLR2andTLR4levels.CandD,silencingTLR2orTLR4reducedcellularBMP-2levelsfollowingstimulation with a receptor agonist for 24 h. E and F, BMP-2 levels were reduced in cells treated with siRNA specific to TLR2 or TLR4 before oxLDL stimulation (80 g/ml, 24h).DensitometrydataareexpressedasmeanS.E.(errorbars).n3;*,p0.05versuscontrol;#,p0.05versuscorrespondenttreatmentwithoutsilencing TLR2 or TLR4.

Article Snippet: Monoclonal neutralizing antibodies to human TLR2 and TLR4 were purchased from Imgenex (San Diego, CA).

Techniques: Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Oxidized Low Density Lipoprotein Induces Bone Morphogenetic Protein-2 in Coronary Artery Endothelial Cells via Toll-like Receptors 2 and 4

doi: 10.1074/jbc.m110.214619

Figure Lengend Snippet: FIGURE 4. Overexpression of TLR2 or TLR4 enhances oxLDL-induced BMP-2 expression. A and B, human CAECs were transfected with plasmids to overexpress TLR2 or TLR4. Representative immunoblots and densitometric data show increased TLR2 and TLR4 levels after transfection. C and D, BMP-2 levels after oxLDL stimulation (80 g/ml, 24 h) were increased in cells overexpressing TLR2 or TLR4. Densitometry data are expressed as mean S.E. (error bars). n 3; *, p 0.05 versus control; #, p 0.05 versus correspondent treatment without overexpressing TLR2 or TLR4.

Article Snippet: Monoclonal neutralizing antibodies to human TLR2 and TLR4 were purchased from Imgenex (San Diego, CA).

Techniques: Over Expression, Expressing, Transfection, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Oxidized Low Density Lipoprotein Induces Bone Morphogenetic Protein-2 in Coronary Artery Endothelial Cells via Toll-like Receptors 2 and 4

doi: 10.1074/jbc.m110.214619

Figure Lengend Snippet: FIGURE 5. OxLDL is co-localized with TLR2 and TLR4 in human CAECs. Cells were treated with oxLDL (80 g/ml, 30 min). Cells were fixed immediately after the treatment. TLR2 or TLR4 was stained green, and oxLDL was stained red by indirect immunostaining. Nuclei were counterstained blue with bis-benzimide. Representative images show that oxLDL is co-localized with TLR2 (A) and TLR4 (B) on cell surfaces (yellow in composite images, arrows).

Article Snippet: Monoclonal neutralizing antibodies to human TLR2 and TLR4 were purchased from Imgenex (San Diego, CA).

Techniques: Staining, Immunostaining