Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: Microscale thermophoresis showing biophysical analysis of binding of human rMBL with human rTLR4/MD2. MST is based on the detection of a temperature-induced change in fluorescence of rTLR4/MD2 (target) as a function of the concentration of a non-fluorescent ligand (rMBL). By titrating MBL into the labeled TLR4 the K d (dissociation constant) was 9.07 × E −07 indicating strong binding.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Microscale Thermophoresis, Binding Assay, Fluorescence, Concentration Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: Effect of human rMBL or human rTLR4 on FD expression on differentiated 3T3-L1 cells at 48 h. (A) rMBL increased FD expression in a dose-dependent manner. (B) rMBL also effected the TLR4 expression. (C) LPS also increased FD expression dose-dependent manner. (D) LPS also decreased TLR4 expression with increasing doses. Data are shown as Mean ± SEM of three replicative experiments. * p < 0.05 considered significant.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

doi: 10.3389/fimmu.2020.00201

Figure Lengend Snippet: A hypothetical model in mouse showing how MBL or conjugates of MBL-MASP-1 or MBL-MASP-2 can regulate the transcription of FD to regulate the activation of the AP via TLR4 receptors. Circulating MBL alone or MBL-MASP-1 or MBL-MASP-2 complexes can directly interact with disguised TLR4 on adipocytes to activate the complement system via enhancing the expression of FD to regulate the AP pathway. (A) MBL or MBL-MASP-1 or MBL-MASP-2 conjugates under normal physiological conditions can bind to the TLR4 and regulate the expression of FD but MASP-2 might be dominant. (B) In contrast, under inflammatory conditions, MBL or MBL-MASP-1 or MBL-MASP-2 conjugates might be displaced by the LPS and modulate the transcription of FD through TLR4 receptors.

Article Snippet: Recombinant Human TLR4 (R&D Systems) was re-suspended in a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , and 1 mM BME.

Techniques: Activation Assay, Expressing

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: International Journal of Molecular Sciences

Article Title: Uncovering the Potential Mechanisms of Ergothioneine in Neuroinflammation Through Network Pharmacology, Molecular Docking, Molecular Dynamics Simulation, and In Vitro Validation

doi: 10.3390/ijms27052179

Figure Lengend Snippet: Representative images of Western blotting of EGT (0.1, 0.5, 1.0 mM) influencing protein expression of p-AKT/AKT, p-PI3K/PI3K, and p-mTOR/mTOR. ( A ) Western blotting of p-AKT/AKT, p-PI3K/PI3K, and p-mTOR/mTOR. ( B ) Quantification of ratio of p-AKT/AKT. ( C ) Quantification of ratio of p-mTOR/mTOR. ( D ) Quantification of ratio of p-PI3K/PI3K. Data are presented as mean ± SEM (n = 3). Values with different letters differ significantly ( p < 0.05).

Article Snippet: The primary antibodies include β-actin (1:5000; Proteintech; Cat. No. 66009-1-lg), NF-κB p65 (1:1000; Proteintech; Cat. No. 10268-1-AP), p-NF-κB p65 (Ser536) (1:1000; CST; Cat. No. 3033), MyD88 (23230-1-AP), TLR4 (19811-1-AP), PI3 Kinase (PI3K) (1:5000; Proteintech; Cat. No. 60225-1-AP), p-PI3K (1:1000; CST; Cat. No. 4228), AKT (1:2000; Proteintech; Cat. No. 10176-1-AP), p-AKT (Ser473) (1:1000; CST; Cat. No. 8326), mTOR (1:5000; Proteintech; Cat. No. 66888-1-AP), and p-mTOR (Ser2448) (1:1000; CST; Cat. No. 5536).

Techniques: Western Blot, Expressing

Journal: Cancer Research

Article Title: ABCB5 Maintains Melanoma-Initiating Cells through a Proinflammatory Cytokine Signaling Circuit

doi: 10.1158/0008-5472.can-14-0582

Figure Lengend Snippet: Figure 5. ABCB5 controls secretion of IL1b, an activator of IL8, to stimulate IL8. A, IL1b secretion by wild-type or DTIC-resistant A375 cells (ELISA). B, IL8 (left) or WFDC1 (right) mRNA expression by A375 WT or patient-derived melanoma cells following exogenous IL1b treatment (tr) versus control (means of n ¼ 3 independentexperiments withthree replicateseach per datapoint; , P < 0.005; , P < 0.0005). C, TLR4expression onABCB5(þ) cells(top) andABCB5()cells (bottom) in G3361 (left) and patient-derived melanoma cells (middle). Aggregate analysis for n ¼ 6 distinct melanoma specimens (G3361, A375, and SK-MEL-28 cell lines, three patient-derived specimens) is shown on the right (, P < 0.05;NS, not significant). D, immunohistochemistry staining for ABCB5 (left,brown), TLR4 (middle, blue), and costaining for ABCB5 and TLR4 (right, purple shows costaining) of a human C8161 melanoma xenograft lung metastasis (left, 400; right, 1,000 magnification). E, left, immunofluorescence costaining of clinical melanoma for TLR4 (red) and ABCB5 (green). (Continued on the following page.)

Article Snippet: Immunofluorescence staining of paraffin-embedded clinical melanoma involved TLR4 antibody (Novus Biologicals) followed by Alexa Fluor594 secondary antibody, and ABCB5 primary antibody (clone 3C2–1D12) followed by addition of biotinylated horse antimouse IgG and Alexa Fluor-488–labeled streptavidin.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Control, Immunohistochemistry, Staining

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