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: 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: 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: 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