16-9917-025

antibody from Invitrogen Antibodies

Targeting: TLR4 ARMD10, CD284, hToll, TLR-4

Provider product page for 16-9917-025

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Antibody data

Antibody Data
Antigen structure
References [42]
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Validations
Flow cytometry [1]
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Product number: 16-9917-025 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD284 (TLR4) Monoclonal Antibody (HTA125), Functional Grade, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The HTA125 monoclonal antibody reacts with human Toll-like receptor 4 (TLR4). So far, at least ten members of the Toll family have been identified in humans. This family of type I transmembrane proteins is characterized by an extracellular domain with leucine-rich repeats and a cytoplasmic domain with homology to the type I IL-1 receptor. Two of these receptors, TLR2 and TLR4, are pattern recognition receptors and signaling molecules in response to bacterial lipoproteins and have been implicated in innate immunity and inflammation. TLR4 physically associates with another molecule called MD-2, and together with CD14, this complex is responsible for LPS recognition and signaling. TLR4 is expressed by peripheral blood monocytes. HTA125 has been reported to immunoprecipitate human TLR4 (~100 kDa) from transfected cells. Most TLR cell surface expression, especially TLR1 and TLR4, occurs at low levels on monocytes and at even lower levels on other cell types including granulocytes and immature dendritic cells (iDC). Furthermore, a relatively high degree of variability in TLR surface expression has been reported among normal donors.
Antibody clone number: HTA125
Concentration: 1 mg/mL

TLR4 protein structure - 16-9917-025 shown in red.

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Experimental details: Figure 2 CD14 and TLR-4 staining in gut epithelial cells. Balb/c mice were not supplemented (control) or supplemented daily by oral gavage with OxC-beta (10 mg/kg). After 4 weeks, intestinal tissues were harvested and CD14 and TLR-4 expression was determined by immunocytochemistry. Increased CD14 (A) and TLR-4 (C) expression is readily apparent in epithelial cells in the OxC-beta-supplemented animals compared to the controls receiving vehicle alone (B and D, respectively). Arrows indicate the location of enterocytes within the cross section of microvilli. Magnification 40x.

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Experimental details: Figure 4 MD-2 is coprecipitated with TLR4. (A) Stable transfectants expressing MD-2 alone or with TLR4 were stained with or without cell permeabilization. The intracellular MD-2 precursor was stained with the anti-protein C mAb. The specificity of the anti-TLR4 mAb was shown by cell surface staining of stable transfectants. Goat anti- mouse IgG-FITC was used as the second reagent. Dotted lines correspond to histograms stained with the second reagent alone. Panels correspond to the original Ba/F3 line, a transfectant expressing TLR4 alone, and a transfectant expressing TLR4 + MD-2, respectively. (B) Stable transfectants expressing TLR4 and MD-2 were subjected to immunoprecipitation with control mouse IgG (lane 1) or the anti-TLR4 mAb, HTA125 (lanes 2 and 3). After blotting, precipitated molecules were detected with an anti-flag mAb, M2. The precipitates shown in lanes 1 and 2 are from the transfectant expressing TLR4 and MD-2, both of which were tagged with the flag epitope. In lane 3, we used the control line in which the flag epitope was on TLR4 but not on MD-2.

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Experimental details: Figure 5 MiR-146-5p Differentially Regulates TLR4, TLR2, and Cytokine Secretion (A) Graphical representation of TLR4 and TLR2 (western blot shown as inserts) in BM-MSCs (n = 6 patient samples) and OM-MSCs (n = 6 patient samples). BM-MSCs have significantly higher levels compared with OM-MSCs (mean +- SEM, * p < 0.05, ** p < 0.01, Students unpaired t test). (B) FACS analysis of TLR4 expression on OM- and BM-MSCs (n = 3, patient samples both). (C) OM-MSC (n = 1 patient sample) and BM-MSC (n = 1 patient sample) cytokine profile. Insert shows dot plot while the graph illustrates the mean pixel intensity of the blot. IL-6 (1) IL-8 (2), and CCL2 (3) were expressed 1.5-fold higher in BM-MSC-CM. (D) ELISA analysis of IL-8, IL-6, and CCL2 in BM-MSC-CM (n = 3 patient samples) and OM-MSC-CM (n = 3 patient samples, mean +- SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, Students unpaired t test).

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Experimental details: Fig. 4 LPS stimulates the expression of SBD-1 through TLR4. a Tlr4 mRNA expression of ovine oviduct stromal cells (OOSCs) and epithelial cells (OOECs) was assessed by qRT-PCR. b PCR was conducted to examine the expression of TLR4 in the ovine oviduct epithelial cells harvested at different times. c Western blotting was performed to examine the expression levels of P38 MAPK after treatment with LPS (100 ng/mL) or a TLR4 neutralizing antibody for 12 h. d The densitometric analysis of the bands on the western blotting showed that LPS could markedly activate P38 MAPK, while the separate addition of the TLR4 neutralizing antibody had no effect. However, treatment with the TLR4 neutralizing antibody could significantly decrease the levels of phosphorylated P38 induced by LPS. e QRT-PCR analysis was used to examine the mRNA levels of SBD-1 after treatment with the TLR4 neutralizing antibody for 12 h. Blocking TLR4 activity could inhibit the expression of SBD-1. All of the experiments were repeated at least three times. * p < 0.05, ** p < 0.01 ( t -test) vs. Control. + p < 0.05, ++ p < 0.01 ( t -test) vs. LPS

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Experimental details: Figure 3 LPS-induced HBD2 expression in A549 cells requires TLR4. A459 cells were incubated with an isotype control or anti-TLR4 neutralizing antibody (Anti-TLR4 mAB 5 mug/ml, 30 min) then, (A) left untreated or stimulated with LPS (10 mug) for 4 or 24 hours. Total RNA was extracted, reverse transcribed into cDNA and used as a template in PCR reactions using HBD2 gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 mug/ml ethidium bromide and visualized under UV. Gels are representative of three independent experiments or (B) left untreated or stimulated with LPS (10 mug) for 24 hours, Fc-blocked and labeled with anti-HBD2 (solid) or isotype control antibodies (clear) and fluorophore-conjugated detection antibodies. HBD2 expression was quantified by laser scanning cytometry, as described, and data from three experiments is presented. HBD2 expression is expressed as Mean Channel Fluorescence (MCF) + SEM. (* P < 0.05 vs control, + P < 0.05 vs control + LPS).

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Experimental details: Fig 6 Circulating histone H2A in plasma from dengue-infected patients activates platelets. ( A-B ) Platelets isolated from healthy volunteers were stimulated with recombinant human histone H2A at the indicated concentrations. ( A ) Platelet surface P-selectin (CD62-P) was evaluated 1, 2 and 4 hours post stimulation by flow cytometry and ( B ) PF4/CXCL4 concentration was measured in supernatants 4 hours post stimulation. ( C-F ) Surface P-selectin and PF4/CXCL4 concentration in the supernatants of platelets stimulated with recombinant histone H2A for 2 hour in the presence of ( C-D ) the calcium chelator BAPTA-AM (20 muM) or vehicle (DMSO); or ( E-F ) blocking antibody against TLR4 (20 mug/mL) or isotype matched IgG. ( G-H ) P-selectin expression on platelets exposed to ( G ) plasma from six dengue-infected patients (dengue plasma) or four heterologous healthy volunteers (control plasma) for the indicated time-points; and ( H ) platelets exposed to dengue plasma or control plasma for 4 hours in the presence of anti-histone H2A (20 mug/mL) or isotype matched IgG. Bars represent mean +- standard error of the mean of 3 independent experiments ( A-F ) and of 4 to 6 independent plasma donors ( G-H ). * indicates p

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Experimental details: Figure 2 BFSE alters phagocytic recognition receptors. MDM were exposure to air control, 1% or 5% BFSE or 10% CSE for 24 h. Cell surface markers: CD36 ( A ), CD44 ( B ), SR-A1 ( C ), CD206 ( D ), TLR-2 ( E ) and TLR-4 ( F ) were detected by immunofluorescence on a FACScanto II flow cytometer and expressed as % positive cells compared to negative controls. (n = 5) *p < 0.05.

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Experimental details: Figure 2. The K694R polymorphism does not affect TLR4 expression levels. (a) Diagram of wild type (WT) and K694R (K694G) TLR4 plasmid vector. (b) mRNA expression levels for the genes TLR4 in human embryonic kidney 293T (HEK293T) cells transiently transfected with WT or K694R TLR4 plasmid or empty vector as control, treated with or without LPS for 6 h. (c) Western blot (WB) analysis of TLR4 expression levels in HEK293T cells transiently transfected with WT, K694R TLR4 plasmid or empty vector treated with or without LPS for 6h. (d) Quantification of TLR4 expression levels in (c). Graphs are the mean +- SD, n = 6, * P < 0.05, Student's t -test.

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Experimental details: Figure 5. The K694R polymorphism does not affect TLR4 interaction with myeloid differentiation factor 2 (MD2). (a) HEK293T cells transiently transfected with WT or K694R TLR4, CD14 and Flag-MD2 plasmids, treated with LPS, were subjected to co-immunoprecipitation with anti-TLR4 Ab, and analysed by WB with anti-Flag Ab. (b) Densitometric quantification of the data shown in (a). The results are presented as ratios of MD2 and TLR4. Graphs are the mean +- SD, n = 3, * P < 0.05, Student's t -test.

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Experimental details: Figure 6. The K694R polymorphism impairs the ability of TLR4 to recruit MyD88. (a) HEK293T cells transiently transfected with WT or K694R TLR4, CD14, MD2 and Flag-MyD88 plasmids, treated with LPS, were subjected to co-immunoprecipitation with anti-TLR4 Ab, and analysed by WB with anti-Flag Ab. (b) Densitometric quantification of the data shown in (a) The results are presented as ratios of MyD88 and TLR4. Graphs are the mean +- SD, n = 3, * P < 0.05, Student's t -test.

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Experimental details: Figure 5 The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 x 10 6 cell/mL were pretreated with 10 mug/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 degC for 30 min and followed by the stimulation with 1 muM HP-NAP, 1 mug/mL Pam 3 CSK 4 , 10 mug/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 degC for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in Figure 2 C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in Figure 2 C and as mean +- SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 x 10 6 cell/mL were pretreated with 10 mug/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 degC for 30 min and followed by the stimulation with 1 muM HP-NAP, 1 mug/mL Pam 3 CSK 4 , 10 mug/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in Figure 2 A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean +- SD of four independe

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Experimental details: Compressive forces and TLR4 blocking antibody modulate TLR4 production and MyD88 in primary PDL cells. ( A ) Protein levels of TLR4 and MyD88 were determined by Western blot in different conditions: HMGB1 (100 ng/mL), TLR4 blocking antibody (5 ug/mL) (TLR4: TLR4 blocking antibody) and compressive force (CF) 2 g/cm 2 for 3 and 24 h. Three different donors showed similar patterns with reduction of TLR4 antibody. TLR4 production was upregulated under 3 h compression forces. MyD88 production was reduced in all conditions. ( B ) Quantification of three donors, normalized to the control with stain-free technology. Data were tested for normal distribution by Shapiro-Wilk test. Afterward, a one-way analysis of variance (ANOVA) followed by Tukeys' post hoc test was performed.

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Experimental details: Compressive forces upregulate phosphor AKT in primary hPDL cells. ( A ) Protein production levels and phospho-AKT were determined by Western blot in different conditions: HMGB1 (100 ng/mL), TLR4 blocking antibody (5 ug/mL) (TLR4: TLR4 blocking antibody) and compressive force (CF) 2 g/cm 2 at 3 and 24 h. Three different donors showed similar patterns. Under all conditions, AKT showed no differences, but the phosphorylated AKT was significantly upregulated with compressive forces (CF) for 3 h and 24 h. Compressive forces with additional blocking TLR4 monoclonal antibody led to significant downregulation comparable to the control. ( B ) Quantification of three donors, normalized to the control with stain-free technology. CF conditions without TLR4 blocking antibody showed a significant upregulation. Data were tested for normal distribution by Shapiro-Wilk test. Afterward, a one-way analysis of variance (ANOVA) followed by Tukeys' post hoc test was performed. Statistically significant differences to control are marked by asterisks (**** p < 0.0001) and hashtag show significant differences between CF and TLR4 +CF (## p < 0.01; #### p < 0.0001).

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Experimental details: Compressive forces upregulate pERK and p P38 in primary hPDL cells. ( A ) Protein levels and phospho-MAPK (ERK, p38 and JNK) were determined by Western blot in different conditions: HMGB1 (100 ng/mL), TLR4 blocking antibody (5 ug/mL) (TLR4: TLR4 blocking antibody) and compressive force 2 g/cm 2 at 3 and 24 h. Three different donors showed similar patterns. Under all conditions, ERK, p38 and JNK showed no differences, but the phosphorylated ERK and p38 were significantly upregulated with compressive forces (CF) as follows: ERK for 3 h and p38 for 3 h and 24 h. ( B ) Quantification of three donors, normalized to the control with stain-free technology. CF conditions without TLR4 blocking antibody showed a significant upregulation for phospho-ERK and phospho-p38. ( C ) HEK-293 and MC3T3 cells were used as a positive control for phospho-JNK antibody. Data were tested for normal distribution by Shapiro-Wilk test. Afterward, a one-way analysis of variance (ANOVA) followed by Tukeys' post hoc test was performed. Statistically significant differences to control are marked by asterisks (* p < 0.05; ** p < 0.01).

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Experimental details: Fluorescence images of CF and TLR4 blocking antibody on PDL cells. ( A ) PDL cells treaded with and without TLR4 blocking antibody (5 ug/mL) and compressive force 2 g/cm 2 for 24 h. NF-kB was stained green (Alexa 488), blue areas represent nuclei (DAPI); scalebar 50 um, CF: compressive force NF-kB was not translocated to the nucleus in any condition. ( B ) Protein production of NF-kB, ERK and phospho-ERK were determined by Western blot. ( C ) Quantification of three donors, normalized to the control with stain-free technology. CF conditions without TLR4 antibody showed a significant upregulation for phospho-ERK and phospho-p38.

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Experimental details: Fig 2 Dot plot and histograms representing TLR4 and TLR2 levels in monocytes. (A1-A3) Monocytes/lymphocyte cell population after CD14-positive cell staining. (B-C) Shift to the right demonstrating an increase in the amount of TLR4 staining in the monocytes. Specific mean fluorescence (MFI) can be quantified from these histograms for each case. (D) TLR4 levels on the population of monocytes as measured using relative mean fluorescence intensity (rMFI). (C) 'reduced EF' patients (EF55%). (E) TLR2 levels on the population of monocytes as measured using rMFI. FACS analysis,* P

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Experimental details: Figure 1. Expression levels of TLR4 and phosphorylation of CRMP2 in the cortex via western blotting. (A) Western blotting was performed to reveal the expression levels of TLR4 and p-CRMP2 in each group. (B) Quantitative analysis of TLR4 expression relative to beta-actin from western blotting as presented in (A). (C) Quantitative analysis of p-CRMP2 expression relative to beta-actin from western blotting a presented in (A). Data are expressed as the mean +- standard deviation (n=5). P

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Experimental details: Figure 4. Butyrate upregulates the levels of TLR4 and CD14 on colon cancer cells. The expression levels of TLR4 and CD14 on the membrane of SW480 cells and CT26 cells treated by butyrate and/or LPS were (A) analyzed using a flow cytometer, and (B) the MFI values of TLR4 and CD14 were quantitative analyzed. These experiments were repeated >=3 times, and representative graphs are presented. Compared with the NC group, *P

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Experimental details: Figure 4 Expression levels of TLR4 and CD14 in senescent and non-senescent endothelial cells. Cell surface expression levels of the LPS receptors TLR4 and CD14 were analyzed with phycoerythrin-conjugated specific antibodies in senescent and non-senescent HUVECs by flow cytometry. (A) Relative expression of TLR4 and (B) CD14 in senescent cells was expressed as a ratio to non-senescent cells. Data are presented as the mean +- SD of at least three independent experiments. * P

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Experimental details: Fig 4 Gene expression of biomarkers of injury and immunostaining of TLR4 in patient auricles. (A-D) TLR4 and NOX4 are activated resulting in elevated TNF-alpha in the auricles. Auricles obtained during CABG surgery presented higher expression of TLR4 (P