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Monoclonal Antibody to human TLR4 (Toll-like receptor 4)/CD284-Azide free

Catalog No	:	IMG-417E
Contents	:	100 ug in 200 ul PBS. Filter (0.2 micron) sterilized. Sodium azide free.
Isotype	:	Mouse IgG2a
Clone	:	HTA125
Purification	:	Protein G Chromatography
Species React	:	Dog, Human
Host	:	Mouse

Application
Flow (Cell Surface): 2-5 ug/ 1x10^6 cells
IP: please see Shimazu et al, 1999 for details
FA (Neutralization): please Basek et al, 2005 for details.
IF/ICC: please see Schneeman et al (2005) for details.
Flow (Intracellular)
Confocal Microscopy: see Scheel et al. (2006) for details
Storage
Store at 4°C, stable for 6 months. For long-term storage, store at -20°C.

Recommended Positive Control : ThP1

Background
The Toll-like receptor (TLR) family in mammal comprises a family of transmembrane proteins characterized by multiple copies of leucine rich repeats in the extracellular domain and IL-1 receptor motif in the cytoplasmic domain. Like its counterparts in Drosophila, TLRs signal through adaptor molecules (1). The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition (2). Ten human homologs of TLRs (TLR1-10) have been described (3). Among this family of receptors, TLR2 and TLR4 have been most studied. These studies have suggested that TLR2 and TLR4 may serve as potential main mediators of LPS signaling (4,5). The TLR4 cDNA codes for a protein consisting of 799 amino acids with approximate molecular weight of 88 kDa (6).

Antigen
This antibody was developed by immunizing mice with Ba/F3 cell line expressing human TLR4 cell surface antigen (9).

Application Notes
The antibody has been shown to block the activation of monocytes with LPS (Paik, et al, 2003). The optimal condition has to be determined for individual experiments.

Genebank Info (Protein)
NP_612564

Reference
1. Muzio M, G Natoli, S Saccani, M Levrero, A Mantovani. 1998. J. Exp. Med. 187: 2097-2101.
2. Medzhitov R, CA Janeway. 1997. Cell 91: 295-298.
3. Chuang TH, RJ Ulevitch. 2001. Biochim. Biophys. Acta 1518: 157-161.
4. Takeuchi O, K Hoshino, T Kawai, H Sanjo, H Takada, K Ogawa, K Takeda, S Akira. 1999. Immunity 11: 443.
5. Poltorak A, P Riccardi-Castagnoli, S Citterio, B Butler. 2000. Proc. Natl. Acad. Sci USA 97: 2163-2167.
6. Medzhitov R, P Preston-Hurlburt P, and CA Janeway Jr. 1997. Nature 388: 394-397.
7. Akashi S, H Ogata, F Kirikae, T Kirikae, K Kawasaki, M Nishijima, R Shimazu, Y Nagai Y, K Fukudome, M Kimoto, K Miyake. 2000. Biochem Biophys Res Commun. 268:172-177.
8. Paik YH, RF Schwabe, R Bataller, MP Russo, C Jobin, DA Brenner. 2003. Hepatology. 5:1043-1055.
9. Shimazu R, S Akashi, H Ogata, Y Nagai, K Fukudome, K Miyake, M Kimoto 1999. MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4. J. Exp. Med. 189: 1777-1782.

Product Citations
1. Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2. Sonja Lotz, Eresso Aga, Inga Wilde, Ger van Zandbergen, Thomas Hartung, Werner Solbach, and Tamás Laskay. J. Leukoc. Biol. 75: 467-477 (2004). Imgenex antibodies cited: 1. TLR2 (IMG-416E) 2. TLR4 (IMG-417) [FA, Fig.7C, (PMN)].
2. NF-kB and C/EBP-driven Interleukin-1 Gene Expression and PAK1-mediated Caspase-1 Activation Play Essential Roles in Interleukin-1 Release from Helicobacter pylori Lipopolysaccharide-stimulated Macrophages. Chaitali Basak, Sushil Kumar Pathak, Asima Bhattacharyya, Debabrata Mandal, Shresh Pathak, and Manikuntala Kundu. J. Biol. Chem. 280: 4279-4288 (2005). Imgenex antibodies cited: 1. TLR4 (IMG-417E) [FA, Fig.7 (THP1 cells)].
3. Evidence of Toll-like receptor molecules on human platelets. Cognasse F, H Hamzeh, P Chavarin, S Acquart, C Genin, and O Garraud. Immunology and Cell Biology 83:196-198. (2205). The following TLR antibodies were cited in this publication: TLR2-PE (IMG-416D), TLR4-PE (IMG-417D), TLR6 (IMG-304A), TLR8-PE (IMG-321D), TLR9-PE (IMG-305D). Applications: Intracellular Flow Cytomety and Cell Surface Flow Cytometry: Figs 1 and 2. A comparison of staining results, intracellular versus cell surface flow cytometry is shown. Cell type: Human platelets
4. Intracellular Signaling Mechanisms Regulating Toll-Like Receptor-Mediated Activation of Eosinophils. Wong CK, PFY Cheung, WK IP and CWK Lam. Am. J. Respir. Cell Mol. Biol.doi:10.1165/rcmb.2006-0457OC (2007), in press. Imgenex antibodies cited (human blood eosinophils and neutrophils from buffy coat): For WB, Fig. 1A: TLR1 (IMG-5012), TLR5 (IMG-664), TLR6 (IMG-304A), TLR7 (IMG-540), TLR8 (IMG-321A), TLR9 (IMG-305A). For Flow (Intracellular) and Flow (Surface), Fig. 1B: TLR1 (IMG-5021), TLR2 (IMG-416C), TLR3 (IMG-315C), TLR4 (IMG-417C), TLR5 (IMG-663C), TLR6 (IMG-304C), TLR7 (IMG-665A), TLR8 (IMG-321C), TLR9 (IMG-305C).
5. Toll-Like Receptor Expression in Human Keratinocytes: Nuclear Factor B Controlled Gene Activation by Staphylococcus aureus is Toll-Like Receptor 2 But Not Toll-Like Receptor 4 or Platelet Activating Factor Receptor Dependent. Martin Mempel, Verena Voelcker, Gabriele Köllisch, Christian Plank, Roland Rad, Markus Gerhard, Christina Schnopp, Peter Fraunberger, Autar K. Walli, Johannes Ring, Dietrich Abeck, and Markus Ollert. J. Invest. Dermatol., 121: 1389-1396 (2003). Imgenex antibodies cited: 1. TLR2 (IMG-416) 2. TLR4 (IMG-417) [IF/ICC, Fig.2A and 2D (human keratinocytes)].
6. Execution of Macrophage Apoptosis by PE_PGRS33 of Mycobacterium tuberculosis Is Mediated by Toll-like Receptor 2-dependent Release of Tumor Necrosis Factor-a. Sanchita B., S. K. Pathak, A. Banerjee, S. Pathak, A. Bhattacharyya, Z. Yang, S. Talarico, M. Kundu, and J. Basu., J. Biol. Chem., 282 1039-1050 (2007). Imgenex antibodies cited: 1. TLR4 (IMG-417A) [FA, Fig.2C (human PBMC)].

7. Cell Activation by Ligands of the Toll-Like Receptor and Interleukin-1 Receptor Family Depends on the Function of the Large-Conductance Potassium Channel MaxiK in Human Macrophages. Scheel O, M. Papavlassopoulos, R. Blunck, A. Gebert, T. Hartung, U. Zahringer, U. Seydel, and AB. Schromm. Infection and Immunity, doi:10.1128/IAI.01783-05 (2006). Imgenex antibodies cited: 1. TLR4 (IMG-417A) [IF/ICC, Fig.3 (HEK293-TLR4/MD2-MaxiK_GFPcells)].
8. RANTES modulates TLR4-induced cytokine secretion in human peripheral blood monocytes. Shahrara S, C Park, V Temkin, J Jarvis, M Volin, R Pope. J. Immunol. 177: 5077-5087 (2006). Imgenex antibodies cited:
1. TLR4 (IMG-417A): Flow (Cell Surface) [PB monocytes], Fig. 2C.
9. 2 TLR2 and TLR4 expression on the immune cells of tuberculous pleural fluid. Prabha C, P Rajashree, D Sulochana. Immunology Letters 117: 26-34 (2008). Imgenex antibodies cited:
1. TLR2- FITC (IMG-416C): Flow (cell surface): Figs. 1A,B (human CD4⁺T cells, CD8⁺T cells, B cells, CD16+56⁺ cells and monocytes); 2(CD4⁺T cells); 4A,B (human Treg cells). Flow (intracellular): Fig. 3A,B (CD4⁺T cells)
2. TLR4- FITC (IMG-417C).Flow (cell surface): Figs. 1B,C (human CD4⁺T cells, CD8⁺T cells, B cells, CD16+56⁺ cells and monocytes); 2(CD4⁺T cells); 4A,B (human Treg cells). Flow (intracellular): Fig. 3A,B (CD4⁺T cells)
10.

Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry. Burgener IA and TW Jungi. Veterinary Immunology and Immunopathology 124:184-191 (2008). Imgenex products cited for canine (dog) PBMC subpopulations:

1. 10083K [IC-Flow (Intracellular Staining Flow Assay) Kit

2. IMG-416A (TLR2), Flow (Cell Surface), Fig 1 and Tables 1 & 2

3. IMG-315A (TLR3), Flow (Intracellular), Tables 1 & 2

4. IMG-417A (TLR4), Flow (Intracellular), Fig 1 and Tables 1 & 2

5. IMG-663A (TLR5), Flow (Cell Surface), Fig 1 and Tables 1 & 2

6. IMG-664A (TLR5), Flow (Cell Surface), Tables 1 & 2

7. IMG-305A (TLR9), Flow (Intracellular), Fig 2 and Tables 1 & 2

11. The probiotic Escherichia coli strain Nissle 1917 induces γς T cell apoptosis via caspase-and FasL-dependent pathways. International Immunology 20:829-840 (2008). Imgenex antibodies cited:
1. IMG-416C (TLR2 FITC) Flow (cell surface), human PBMCs, Fig. 5.
2. IMG-417C (TLR4 FITC) Flow (cell surface), human PBMCs, Fig. 5.
12. Pro-osteogenic phenotype of human aortic value interstitial cells is associated with higher levels of toll-like receptors 2 and 4 and enhanced expression of bone morphogenetic protein 2. Yang X, D Fullerton, X Su, L Ao, J Cleveland, X Meng. Journal of the American College of Cardiology 53: 491-500 (2009). Imgenex antibodies cited:
1. IMG-416D (TLR2-PE): WB, Flow (cell surface): human aortic valve interstitial cells, Figs. 1A,B; 2C; 6A.
2. IMG-417D (TLR4-PE): WB, Flow (cell surface): human aortic valve interstitial cells, Figs. 1A,B; 2C; 6A.
13.

Stimulation of iNOS expression and apoptosis resistance in B-cell chronic lymphocytic leukemia (B-CLL) cells through engagement of Toll-like receptor 7 (TLR-7) and NF-kB activation. Hammadi A, Billard C, A-M Faussat, J-P Kolb. Nitric Oxide 19:138-145 (2008).Imgenex products cited (B-CLL cells from a leukemia patient):

1. IMG-417E (TLR4-FITC, clone HTA125): Flow (Intracellular), Fig 1: TLR4 was undetectable in the B-CLL cells.

2. IMG-305C (TLR9-FITC, clone 26C593.2): Flow (intracellular), Fig 1: TLR9 was detected in the BCLL cells

3. IMK-501 (Phospho-IkBa ActivELISA Kit): Fig 6B, ELISA data shows increased of IkBa phosphoryation following resiquimod treatment which stimulates NF-kB activation.

Note: No expression of TLR-4 or TLR-9 was detected at the surface of B-CLL cells by Flow (cell surface).

14. Functional heterogeneity and differential priming of circulating neutrophils in human experimental endotoxemia. Pillay J, B Ramaker, V Kamp, A Loi, S Lam, F Hietbrink, L Leenen, A Tool, P Pickkers, L Koenderman. J Leuko Bio doi:10.1189/jlb.1209793
TLR4 (IMG-417A): Flow (cell surface), human neutrophils and leukocytes, Fig 2.
15. High dose of bacterial LPS induce apoptosis-dependent tight junctional destruction in intestinal epithelial cells: Role of CD14. Wei-ting K, L Yu. http://ntur.lib.ntu.edu.tw/handle/246246/17800 (2008). The following monoclonal antibodies were used in flow (cell surface) cytometry: TLR4/CD284 (IMG-5031A) & TLR4/CD284 (IMG-417A).
TLR4 was detected in T84 human colonadenocarcinoma cells but was not expressed in Caco-2 human colon carcinoma cells, various figures.
16. Human lymphatic endothelial cells express multiple functional TLRs. Pegu A, S Qin, B Junecko, R Nisato, M Pepper, T Reinhart. J Immunol 180: 3399-3405 (2008). The following antibodies were used in flow (cell surface and intracellular) cytometry: TLR2/CD282 PE (IMG-416D), TLR4 (IMG-417D), TLR5 FITC (IMG-663C), TLR6/CD286 FITC (IMG-304C), TLR9/CD289 PE (IMG-305D). Primary human lung microvascular endothelial cells, Fig 1C & 1D.

Note: Data described but not shown for TLR9/CD289 PE (IMG-305D).
17. Toll like receptor expression and function in subsets of human T lymphocytes. Pietschmann K, S Beetz, S Welte, I Martens, J Gruen, H Oberg, D Wesch, D Kabelitz. Scandinavian J Immunol 70: 245-255 (2009).
1. TLR4/CD284 (IMG-417A): Flow (cell surface), Human T cell subsets (V sigma 1 and V sigma 2) isolated from PBMCs, Fig 3.
2. TLR6/CD286 (IMG-304A) & TLR8/CD288 PE (IMG-321D): Flow (intracellular), Human T cell subsets (V sigma 1 and V sigma 2) isolated from PBMCs, Fig 3.
18. In vitro response to Candida albicans in cultures of whole human blood from young and aged donors. Murciano C, E Vilamon, A Yanez, J Murciano, A Mir, D Gozalbo, M Gil. Immunol & Med Microbiol 51: 327-335 (2007). The following anitbodies were used in flow (cell surface) cytometry: TLR2/CD282 FITC (IMG-416C), TLR4 FITC (IMG-417C), TLR6/CD286 FITC (IMG-304C). Human whole blood from both young and elderly volunteers, Fig 1.

Note: The fluorescence mean intensity was measured in lymphocytes, monocytes, granulocytes (gated by their respective properties in forward and side scatter), Fig 1.
19. TLR4 dependent induction of vascular adhesion molecule-1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2alpha/Cyclooxygenase-2. Wu C, P Chi, H Hsieh, S Luo, C Yang. J Cell Physiol 223:480-491 (2010). IMGENEX product cited: TLR4 (IMG-417E). WB: Human rheumatoid arthritis synovial fibroblasts treated with LPS, Fig 1.
20. TAK-242 (Resatorvid), a small molecule inhibitor of toll like receptor 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules. Matsunaga N, N Tsuchimori, T Matsumoto, M Li. Mol Pharma 79:34-41 (2011). IMGENEX product cited: TLR4/CD284 (IMG-417A). IP: HEK293 cells were immunoprecipitated with TLR4 (IMG-417A) and WB with a FLAG antibody, Fig 4B.
21. MD-2, a molecule that confers lipopolysaccharide responsiveness on toll like receptor 4. Shimazu R, S Akashi, H Ogata, Y Nagai, K Fukudome, K Miyake, M Kimoto. IMGENEX product cited: TLR4/CD284 (IMG-417A). WB: Human cell lines: Nalm-6, Ramos, Daudi, RPMI8866, CEM, Molt4, U937, THP-1, and K562 (erythroleukemia) , Fig 1A.
22. HIV-1 Tat protein suppresses cholangiocyte toll like receptor 4 expression and defense against Cryptosporidium parvum. O'hara S, A Small, G Gajdos, A Badley, X Chen, N LaRusso. J Infect Dis 199:1195-1204 (2009). IMGENEX product cited: TLR4/CD284 (IMG-417A). WB: Human intestinal (HT-29) lysate infected with C. parvum, Fig 2.
IF: C. parvum infected cholangiocytes, Fig 2.
23. A molecule that is associated with Toll-like receptor 4 and regulates its cell surface expression. Konno K, Y Wakabayashi, S Akashi-Takamura, T Ishii, M Kobayashi, K Takahasi, Y Kusumoto, S-i Saitoh, Y Yoshizawa, K Miyake. BBRC 339:1076-1082 (2006). Imgenex TLR4 antibodies cited:
1. IMG-428A (TLR4, Clone MTS510): IP (Figs 3 & 4), TLR4-transfected Ba/F3 cells. The antibody immunoprecipitated both the immature (less glycosylated) and mature (more glycosylated) forms of TLR4.

2. IMG-417A (TLR4, Clone HTA125): Flow (Cell Surface) (Fig 5B), TLR4-transfected HEK293 cells.

Note: The specificity of the IMG-428A and IMG-417A TLR4 mAbs was validated in TLR4-transfected cells by IP (Figs 3 & 4) and Flow (Cell Surface) (Fig 5), respectively.
24. In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes. Giovanna Zanoni, Riccardo Navone, Claudio Lunardi, Giuseppe Tridente, Caterina Bason, Simona Sivori, Ruggero Beri, Marzia Dolcino, Enrico Valletta, Roberto Corrocher, Antonio Puccetti. PLoS Med 3(9): e358. DOI: 10.1371/journal.pmed.0030358 (2006). Imgenex antibodies cited- The anti-TLR4 monoclonal antibody clone HTA125, isotype IgG2b (Imgenex) was used..

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