Anti tlr4

Manufactured by Novus Biologicals

Sourced in United States, United Kingdom

Anti-TLR4 is a laboratory reagent designed to detect and/or study the Toll-like Receptor 4 (TLR4) protein. TLR4 is a member of the Toll-like receptor family, which plays a crucial role in the innate immune response. This product can be used in various in vitro research applications to investigate the expression, function, and regulation of TLR4.

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Spelling variants of this product

Anti-TLR4 antibody (4 citations) Rabbit anti-TLR4 (3 citations) Anti-TLR4 (NB-10056566, (2 citations)

14 protocols using anti tlr4

Immunohistochemical Profiling of BAMBI and TLR4 in Prostate

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For immunohistochemical staining of prostate specimens, paraffin sections were deparaffinized and rehydrated. The sections were incubated with 3% hydrogen peroxide solution for ∼12 min and then incubated in a blocking solution containing 5% bovine serum albumin for 30 min. Next, the sections were incubated with anti-BAMBI (1:100; Cell Signalling Technology; USA) and anti-TLR4 (1:100; Novus; USA) antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP). The secondary antibody (1:100; Sigma-Aldrich, USA) was incubated at 25–28 °C. Images were obtained using a microscope (Nikon, Tokyo, Japan).
BPH-1 cells were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, the cells were permeabilized with 0.5% Triton X-100 for 15 min. Briefly, the endogenous peroxidase activity of the cells was blocked by treatment with 3% H₂O₂/PBS for 20 min. They were subsequently blocked with 0.2% fish gelatine for 20 min and incubated with primary antibodies, anti-TLR4 (1:100; Novus, USA), and anti-BAMBI (1:100; Cell Signalling Technology; USA), overnight at 4 °C. After washing, the cells were incubated with secondary antibodies for 60 min and nuclear staining was performed. Images were acquired using a fluorescence microscope (Nikon, Tokyo, Japan).

Liu Y., Wang Z., Gan Y., Chen X., Zhang B., Chen Z., Liu P., Li B., Ru F, & He Y. (2021). Curcumin attenuates prostatic hyperplasia caused by inflammation via up-regulation of bone morphogenetic protein and activin membrane-bound inhibitor. Pharmaceutical Biology, 59(1), 1024-1033.

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TLR2/TLR4 Involvement in AEAR-activated DCs

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To determine whether TLR2 or TLR4 was involved in the AEAR-induced DCs activation, DCs were pre-incubated with anti-TLR2 (5 μg/mL) or anti-TLR4 (2 μg/mL) (Novus Biologicals, Littleton, CO, USA) for 1 h, and then AEAR (50 or 400 μg/mL) was added for 12 h incubation. The expression levels of CD40, CD86, IL-1β, and IL-6 were measured as described above.

Yang Y., Wang D., Li Q., He J., Wang B., Li J, & Zhang A. (2020). Immune-Enhancing Activity of Aqueous Extracts from Artemisia rupestris L. via MAPK and NF-kB Pathways of TLR4/TLR2 Downstream in Dendritic Cells. Vaccines, 8(3), 525.

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Quantitative Analysis of Innate Immune Signaling

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Mφ transfected with either Mal siRNA or control siRNA were washed with PBS and disrupted with lysis buffer (50 mmol L⁻¹ Tris-HCl, 250 mmol L⁻¹ NaCl, 5 mmol L⁻¹ EDTA, 1 mmol L⁻¹ dithiothreitol, 5% glycerol, 0.2% Nonidet P-40 at pH 8) containing a protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of whole cell lysates (50–100 μg) were loaded in each lane for separation by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking in Tris-buffered saline containing Tween 20 (0.1%) (T-TBS) and milk (5%) at room temperature for 2 h, the membrane was then incubated at 4°C overnight with primary antibodies. After washing with T-TBS, the membrane was incubated at room temperature for 1 h with HRP-conjugated anti-rabbit IgG secondary antibody (Cell Signaling Technology, Danvers, MA). The primary anti-Mal (Abcam, Cambridge, UK), anti-MyD88 (Cell Signaling Technology), anti-TRIF (Novus Biologicals, Littleton, CO), anti-TLR-4 (Novus Biologicals) and anti-β-actin (Sigma- Aldrich) antibodies were diluted and used as described in the manufacturer’s instructions. The density of each band was quantified by densitometric analysis with Image J software (Image Processing and Analysis in Java) from the NIH (https://imagej.nih.gov/ij/).

Zhou S., Qi Q., Wang X., Zhang L., Xu L., Dong L., Zhu J., Li Y., Wang X., Xu Z., Liu F., Hu W., Zhou L., Chen X, & Su C. (2018). SjHSP60 induces CD4+CD25+Foxp3+ Tregs via TLR4-Mal-drived production of TGF-β in macrophages. Immunology and cell biology, 96(9), 958-968.

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Toll-like Receptor Protein Expression Analysis

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The cells were washed with PBS and the lysates were prepared as previously described (16 (link)). Protein quantification was performed using a DC^TM protein assay kit (Bio-Rad) and run on SDS-PAGE gels. The primary antibodies consisted of anti-TLR1, anti-TLR2 (R&D Systems), anti-TLR4 (Novus Biologicals), anti-TLR10 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-IκBα #9246 (Cell Signaling), NF-κBα p65 sc-372 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin antibodies. HRP-labeled goat anti-mouse IgG 1706516 (Bio-Rad) and HRP-labeled goat anti-rabbit IgG 1706515 (Bio-Rad) were used as secondary antibodies.

Henrick B.M., Yao X.D., Zahoor M.A., Abimiku A., Osawe S, & Rosenthal K.L. (2019). TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection. Frontiers in Immunology, 10, 482.

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NLRP3, Caspase-1, and TLR-4 Protein Analysis

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Expressions of NLRP3, caspase-1 and TLR-4 levels were studied by WB analysis. Protein was extracted from total tissues and washed in PBS, suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM DTT, 0.1% SDS and 1 mM PMSF, and placed on ice for 30 mins. After centrifugation for 15 mins at 4°C, the supernatant was collected. Protein concentrations were determined using a BCA assay (Pierce, Rockford, IL, USA). For WB analysis, equal amounts of proteins (50 µg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to aThe membrane was blocked using blocking buffer (5% non-fat dry milk and 1% Tween-20 in PBS) for 1 hr at room temperature, then incubated with appropriate primary antibodies: anti-NLRP3 (Novus Biologicals, USA) (1:500), anti-caspase-1 (CST, USA) (1:1,000) and anti-TLR4 (Novus Biologicals, USA) (1:1,000) in blocking buffer overnight at 4°C. The blot was then incubated with appropriate alkaline phosphatase (AP)-conjugated secondary antibody: anti-rabbit IgG, HRP-linked antibody (CST, USA) (1:2,000) and detected in 5 mL AP buffer containing 16.5 µL BCIP and 33 µL NBT at room temperature for 10–20 mins, then photographed. GAPDH (KC, People's
Republic of China) (1:5,000) was used as a loading control.

Chen H., Ding Y., Chen W., Feng Y, & Shi G. (2019). Glibenclamide alleviates inflammation in oleic acid model of acute lung injury through NLRP3 inflammasome signaling pathway. Drug Design, Development and Therapy, 13, 1545-1554.

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Characterization of Pyrogenic Silica Nanoparticles

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Pyrogenic amorphous silica nanoparticles (ASNP) NM-203 were from the same batch used in [19 (link)] and were obtained from the JRC Nanomaterials Repository (Ispra, Varese, Italy). The physico-chemical characterization of NM-203 ASNPs, before and after their suspension in different media, has been extensively reported elsewhere [17 (link)]. A/B Human serum and E. coli LPS (strain O55:B5) were from Sigma-Aldrich (Merck, Milan, Italy), while RPMI 1640 culture medium was provided by Corning (New York, NY, USA). The antibodies used were mouse monoclonal anti-GS (BD, Franklin Lakes, NJ, USA), rabbit polyclonal anti-LC3BI/II, rabbit polyclonal anti-p-NFκB, mouse monoclonal anti-NFκB, mouse monoclonal anti-LAMP1 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-TLR4 (Novus Biologicals, Cambridge, UK). All antibodies were diluted 1:1000 unless otherwise specified.

Bianchi M.G., Chiu M., Taurino G., Bergamaschi E., Cubadda F., Macaluso G.M, & Bussolati O. (2022). The TLR4/NFκB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles. Nanomaterials, 12(13), 2307.

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Multicolor Flow Cytometry of Gut Lymphocytes

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For flow cytometric analysis, 5x10⁵ PPL, IEL and LPL were labelled with mouse anti-rat monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PerCP) or allophycocyanin (APC). The antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25 (BD Biosciences, Oxford, UK), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA) and anti-TLR4 (Novus Biologicals, Littleton, CO, USA). The cells were stained as previously described [19 (link)]. Briefly, lymphocytes were incubated with saturating amounts of antibodies in PBS-0.2% FBS-0.1% NaN₃ (darkness, 20 min, 4 °C). Consecutively, the cells were washed, and fixed with 0.5% p-formaldehyde (darkness, until analysis, 4 °C). A negative control staining was included in each cell sample. Analyses were performed with a Gallios Cytometer (Beckman Coulter, Miami, FL, USA) in the Scientific and Technological Centres of the University of Barcelona (CCiTUB).

Camps-Bossacoma M., Pérez-Cano F.J., Franch À., Untersmayr E, & Castell M. (2017). Effect of a cocoa diet on the small intestine and gut-associated lymphoid tissue composition in an oral sensitization model in rats. The Journal of nutritional biochemistry, 42, 182-193.

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Comprehensive Flow Cytometric Analysis

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For flow cytometric analysis, splenocytes (2 × 10⁵) were labelled with mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-Cyanine (Cy)7, as in previous studies^{60 (link)}. In this case, the antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA), anti-TLR-4 (Novus Biologicals, Littlon, CO, USA) and anti-Foxp3 (eBioscience). After staining with standard procedures^{20 (link)}, analyses were performed using a Gallios^TM Cytometer (Beckman Coulter Inc., Miami, FL, USA) at the CCiT-UB. All results were assessed by the Flowjo v.10 software (TreeStar, Inc., Ashland, OR, USA).

Grases-Pintó B., Abril-Gil M., Castell M., Pérez-Cano F.J, & Franch À. (2019). Enhancement of immune maturation in suckling rats by leptin and adiponectin supplementation. Scientific Reports, 9, 1786.

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Organic Synthesis and Cell Signaling Assays

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Chemicals used for organic synthesis were purchased from commercial sources and used without purification except when otherwise stated. ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) spectra were recorded on a Bruker 600 instrument. Chemical shifts were reported in ppm and referenced to the residue of solvent: (CDCl₃: 7.26 ppm for ¹H; 77.0 ppm for ¹³C). Melting points were determined on Fargo MP-2D and not corrected. HRMS were recorded on Finnigan MAT-95S. Dulbecco’s modified Eagle’s medium (DMEM), lipopolysaccharide (LPS), and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were obtained from Biowest (Kancas, MO, USA). Anti-TLR4 (rabbit anti-toll-like receptor 4 polyclonal antibody), anti-COX-2 (rabbit anti-cyclooxygenase-2 polyclonal antibody), and anti-β-actin (mouse anti-beta actin monoclonal antibody) antibodies were purchased from Novus (Littleton, CO, USA). Peroxidase-conjugated sheep anti-mouse IgG and goat anti-rabbit IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA).

Shih T.L., Liu M.H., Li C.W, & Kuo C.F. (2018). Halo-Substituted Chalcones and Azachalcones Inhibited Lipopolysaccharited-Stimulated Pro-Inflammatory Responses through the TLR4-Mediated Pathway. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(3), 597.

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Immunohistochemical Analysis of Inflammatory Markers

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After antigen retrieval at a high pH for 20 min, paraffin-embedded sections were incubated with anti-TLR-4 (1:100; Novus Biologicals, LLC), anti-GAL-3 (1:100; Novus Biologicals, LLC), anti-MIF (1:200; Abcam, Cambridge, UK), anti-CD45 (1:100; Abcam, Cambridge, UK), anti-CD68 (1:100; Abcam, Cambridge, UK), anti-vimentin (1:200; Cell Signaling Technology, Inc.), and anti-fibroblast growth factor-basic (1:500; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The sections were then washed with Tris-buffered saline and incubated with the biotinylated secondary antibody Histofine Simple Stain Rat MAX PO (Nichirei Bioscience, Nichirei Corporation, Tokyo, Japan.), and the diaminobenzidine substrate was added to react with horseradish peroxidase (HRP) following the protocol of Dako REAL Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako Products, Santa Clara, CA, USA). Finally, the sections were stained with hematoxylin. Semiquantitative analysis of immunostained slides was performed using Image-Pro Plus Version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Wu Z.S., Lo J.J., Wu S.H., Wang C.Z., Chen R.F., Lee S.S., Chai C.Y, & Huang S.H. (2018). Early Hyperbaric Oxygen Treatment Attenuates Burn-Induced Neuroinflammation by Inhibiting the Galectin-3-Dependent Toll-Like Receptor-4 Pathway in a Rat Model. International Journal of Molecular Sciences, 19(8), 2195.

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