14-9917-80

antibody from Invitrogen Antibodies

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

Provider product page for 14-9917-80

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Blocking/Neutralizing

Other assay

Antibody data

Antibody Data
Antigen structure
References [101]
Comments [0]
Validations
Flow cytometry [1]
Other assay [30]

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Product number: 14-9917-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD284 (TLR4) Monoclonal Antibody (HTA125), 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. It is highly recommended that for optimal staining of TLR4, whole blood be stained using the lysed whole blood protocol (found in Best Protocols) rather than Ficoll-gradient prepared normal human peripheral blood cells. The use of a density gradient appears to reduce the staining intensity significantly. Applications Reported: The HTA125 antibody has been reported for use in flow cytometric analysis, and immunoprecipitation. It has also been reported in blocking of LPS-induced cytokine production. For detection of peripheral monocytes, a three step staining protocol is recommended using purified anti-human TLR4 followed by biotin anti-mouse IgG and streptavidin-PE. (Please use Functional Grade purified HTA125, Product # 16-9917-82, in functional assays). Applications Tested: The HTA125 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Purity: Greater than 90%, as determined by SDS-PAGE. Aggregation: Less than 10%, as determined by HPLC. Filtration: 0.2 µm post-manufacturing filtered.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: HTA125
Vial size: 25 µg
Concentration: 0.5 mg/mL
Storage: 4° C

TLR4 protein structure - 14-9917-80 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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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.

Supportive validation

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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

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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).

Supportive validation

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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).

Supportive validation

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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.

Supportive validation

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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

Supportive validation

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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

Supportive validation

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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

Supportive validation

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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

Supportive validation

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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