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Monoclonal Antibody to TLR3 (Toll-like receptor 3)/CD283 Alexa Fluor® 488 Conjugate
Catalog No : IMG-315AF488
Contents : 0.1 mg antibody in 0.2 ml PBS containing 0.05% BSA and 0.05% sodium azide. Sodium azide is highly toxic.
Isotype : Mouse IgG1, Kappa
Clone : 40C1285.6
Purification : Protein G Chromatography
Species React : Dog, Human, Mouse
Host : Mouse
Application
Flow (Intracellular): 0.5-1 ug/10^6 cells 
Flow (Cell Surface): see Wong et al, 2007 for details
IF/ICC: see Ranjith-Kumar et al, 2007 for details.
Storage
Product is light- and temperature-sensitive.  Store antibody in the dark at 4°C.  Do not freeze.

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, TLR proteins signal through adaptor molecules (1) and could constitute an important and unrecognized component of innate immunity in humans. The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition (2). TLR proteins characterized so far activate the MyD88/interleukin-1 receptor-associated kinase (IRAK) signaling pathway. Ten human homologs of TLR proteins (TLR1-10) have been described (3). TLR3 cDNA codes for a protein with approximate molecular weight of 120 kDa (4). TLR3 has a restricted expression pattern being expressed in dendritic cells (DC) (4-6). TLR3 mRNA expression was detected by in situ hybridization in DC and lymph nodes (6). The expression of TLR3 in a single cell type may indicate a specific role for this molecule in a restricted setting.



Antigen
This antibody was developed against KLH-conjugated synthetic peptide of human TLR3.

Intracellular flow cytometric analysis of TLR3 in human monocytes probed with 1 ug/10^6 cells of AF488-conjugated TLR3 antibody. Shaded histogram represents cells without antibody; green represents isotype control antibody (IMGENEX, 20108); red represents TLR3 antibody.

Flow cytometric analyis of CD14-/CD11c+/TLR3+ myeloid dendritic cells (mDCs). Cells were stained for surface markers CD14 (IMGENEX, IMG-6350D) and CD11c, and intracellular stained for TLR3. CD14- cells were gated and stained with AF488-conjugated isotype control antibody (IMGENEX, 20108), and APC-conjugated CD11c antibody (left). CD14- cells were gated and stained with 1 ug AF488-conjugated TLR3 antibody (IMGENEX, IMG-315AF488), and APC-conjugated CD11c antibody (right).

Related Products
1. 10083K [IC-Flow (Intracellular Staining Flow Assay) Kit]
2. IMG-2203 [Poly(I).Poly(C), TLR3 ligand]

Reference
1. Muzio M, Natoli G, Saccani S, Levrero M, and Mantovani A. J. Exp. Med. 187: 2097-2101 (1998).
2. Medzhitov R and Janeway CA. Cell 91: 295-298 (1997).
3. Chuang TH and Ulevitch RJ. Biochim. Biophys. Acta 1518 (1-2): 157-161 (2001)
4. Rock FL., Hardiman G., Timans JC., Kastelein RA. and Bazan JF. Proc. Natl. Acad. Sci. U.S.A. 95 (2), 588-593 (1998).
5. Muzio M, Polentarutti N, Bosisio D, Prahladan MK, Mantovani A. J Leukoc Biol 67(4):450-456 (2000).
6. Muzio M, Bosisio D, Polentarutti N, D'amico G, Stoppacciaro A, Mancinelli R, van't Veer C, Penton-Rol G, Ruco LP, Allavena P, Mantovani A. J Immunol 164(11):5998-6004 (2000).

Product Citations
1. Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain. C.T. Ranjith-Kumar, W Miller, J Xiong, W K. Russell, R Lamb, J Santos, K E. Duffy, L Cleveland, M Park, K Bhardwaj, Z Wu, D H. Russell, R T. Sarisky, M Lamine Mbow, and C. Cheng Kao. J. Bio. Chem., 282 7668-7678 (2007). Imgenex antibodies cited: 1. TLR3 (IMG-315A) [WB, Fig1A, Fig3B, Fig4D, Fig5D, Fig7B (human TRL3 transfected cells)] 2. TLR3 (IMG-315C) [IF, Fig3C, Fig4E, Fig5E, Fig7C, Fig9D (human TLR3 transfected cells)].
2. 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).
3. Toll-like receptors in cellular subsets of human tonsil T cells: altered expression during recurrent tonsillitis. Mansson A, M Adner and LO Cardell. Respiratory Resesarch doi:10.1186/1465-9921-7-36. (2006).  Imgenex antibodies cited (human tonsils separated into cell subtypes): 1. TLR3 [IMG-315D (Flow-Intracellular), Figs 5 and 6]. 2. TLR5 [IMG-663A (Flow-Intracellular), Fig 6]. 3. TLR9 [IMG-305C (Flow-Intracellular), Fig 4.].
4. High-avidity antitumor T-cell generation by toll receptor 8-primed, myeloid-derived dendritic cells is mediated by IL-12 production. Xu S., U. Koldovsky, M. Xu, D. Wang, E. Fitzpatrick, G. Son, G. Koski, and B. J. Czerniecki., Surgery, 140:170-178 (2006). Imgenex antibodies cited: 1. TLR2 (IMG-416) 2. TLR3 (IMG-315) 3. TLR4 (IMG-417) 4. TLR8 (IMG-321) [Flow (cell surface and intracellular), Fig.1A and 1B (human monocytes)].
5. Inhibition of neutrophil apoptosis by TLR agonists in whole blood: involvement of the phosphoinositide 3-Kinase/Akt and NF-kB signaling pathways, leading to increased levels of Mcl-1, A1, and phosphorylated bad.  François S, J El Benna, M Pham . C Dang, E Pedruzzi, M Gougerot-Pocidalo, C Elbim. J. Immunol 174: 3633-3642 (2005). Imgenex antibodies cited [WB (human polymorphonuclear neutrophils (PMN)), Fig. 5]:
1. TLR2 (IMG-319)
2. TLR3 (IMG-315A)
3. TLR5 (IMG-664)
4. TLR7(IMG-581A)

6. STAT3 and STAT5-dependent pathways competittively regulate the pan-differentiation of DC34pos cells into tumor-competent dendritic cells. Cohen P, Koski G, Czerniecki B, Bunting K, Fu X, Wang Z, Zhang W, Carter C, Awad M, Distel C, Nagem H, Paustian C, Johnson T, Tisdale J, Shu S. Immunobiology 112: 1832-1843 (2008). Imgenex antibodies citied [Flow (intracellular), mouse bone marrow cells, Fig. 1E]:
1. TLR3 FITC (IMG-315C)
2. TLR4 FITC (IMG-417C)
3. TLR7 (IMG-665A)
4. TLR8 FITC (IMG-321C)
5. TLR9 FITC (IMG-305C)

7.

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

8. The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling. Funami K, M Matsumoto, H Oshiumi, T Akazawa, A Yamamoto, and T Seya. InternationalImmunology 16:1143-1154 (2004).  Antibody format cited for WB: IMG-315A (HEK293 cells transfected with various TLR3 plasmid constructs), Fig 3A and 5C,D,F. Note: The IMG-315A antibody is transfected validated in this study. It is also shown to WB TLR3 that has been immunoprecipitated with another TLR3 antibody, Clone TLR3.7 generated in the investigator's laboratory.
9.
Down modulation of human TLR3 function by a monoclonal antibody. Duffy KE, RJ Lamb, LR San Mateo, JL Jordan, G Canziani, M Brigham-Burke, J Korteweg, M Cunningham, HS  Beck, J Carton, J Giles-Komar, C Duchala, RT Sarisky, ML Mbow. Cellular immunology 248:103-114 (2007).Antibody format cited for WB: IMG-315A (recombinant extracellular domain of human TLR3), Fig 1B. Note: IMG-315A recognized recombinant TLR3 (125 ng) when processed under both denaturing (Fig 1B left) and native (Fig 1B right) conditions. 

10. Engagement of TLR2 reverses the suppressor function of conjunctiva CD4+CD25+ regulatory T cells, and promotes herpes simplex virus epitope-specific CD4+CD25 effector T cell responses. Dasgupta G, A Chentoufi, S You, P Falatoonzadeh, L Urbano, A Akhtarmalik, K Nguyen, L Ablabutyan, A Nesburn, L BenMohamed. IOVS doi:10.1167/iovs.10-6522. (2011). IMGENEX products cited:
Flow (intracellular), Fig 1A ( CD4+CD25+ regulatory T cells purified from rabbit conjunctiva) & Fig 2A (CD11b+ cells isolated from rabbit conjunctiva):  TLR2/CD282 PE (IMG-416D), TLR3/CD283 (IMG-315D), TLR4/CD284 (IMG-417D), TLR8/CD288 (IMG-321D), TLR9/CD289 (IMG-305D).

FA, Fig 7 (CD4+CD25+ Treg cells): Pam3CSK4 [TLR1 and TLR2 ligand (IMG-2201)], Pam3CSK4 treated and untreated cells were analyzed by Flow (intracellular).

11. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. Kuznik A, M Bencina, U Svajger, M Jeras, B Rozman, R Jerala. J Immunol 186:4794-4804 (2011). IMGENEX product cited: TLR3/CD283 (IMG-315A). WB: HEK293 cells in the presence of quinacrine or choloroquine, Fig 2E.
12. Toll like receptor agonists induce inflammation and cell death in a model of head and neck squamous cell carcinomas. Immunology 128:e600-e611 (2009). IMGENEX products cited: TLR2/CD282 PE (IMG-416D), TLR3/CD283 (IMG-315A), TLR3/CD283 (IMG-315D), TLR5 PE (IMG-663D), TLR5 (IMG-663).

1. IHC(paraffin): Human head and neck squamous (HNSCC) cell carcinomas showing keratinized cell carcinoma from the larynx stained with TLR2 (IMG-416A) and TLR5 antibodies (IMG-663A), Fig 1A,1B, & 1C.
2. Flow (intracellular): TLR2 PE (IMG-416D), TLR3 PE (IMG-315D), and TLR5 PE (IMG-663D) antibodies were used in human bronchial epithelial (NL-20) and human pharyngeal squamous (Detroit-562) carcinoma cell line, Fig 2C.

13.
The role of UNC93B1 protein in surface localization of TLR3 receptor and in cell priming to nucleic acid agonists. Pohar J, N Pirher, M Benčina, M Manček-Keber and R Jerala. J Biol Chem 288: 442-454 (2013). IMGENEX antibodies cited: TLR3 (IMG-315A) for WB:
Fig 1E: HUVEC cells,  TLR3 was detected at ~130 kDa. IFN-Beta treatment upregulated TLR3 expression and induced TLR3 glycosylation (TLR3 form higher than 130 kDa).
 
Fig 4B, C (TLR3 transfected HEK293T). When UNC93B1 was over-expressed with TLR3, TLR3 glycosylation was increased and a TLR3 form higher than 130 kDa was observed.  UNC93B1 expression also led to upregulation of TLR3 cell surface expression.
 
Fig 5C, F (TLR3 transfected HEK293T).
 
Note: The specificity of the antibody was validated in TLR3 transfected HEK293T by WB (Figs 4B, 4C, 5C, 5F). 14. Characterization of the Toll-like receptor expression profile in human multiple myeloma cells. Abdi J, T Mutis, J Garssen, F Redegeld. PLoS ONE 8(4): e60671. doi:10.1371/journal.pone.0060671 (2013). IMGENEX products cited: TLR1 (IMG-5012), TLR2 (IMG-416A), TLR3 (IMG-315A), TLR4 (IMG-5031A), TLR5 (IMG-663A), TLR7 (IMG-581A), TLR8 (IMG-321A) and TLR9 (IMG-305E).15. Toll-Like receptor 3 antagonists for the treatment of metabolic and cardiovascular diseases.Cunningham M, Y Feng K Heeringa, J Luo, R Rauchenberger, M Rutz, L San Mateo, R Sarisky, V Stojanovic-susulic, R Sweet, F Teng, A Teplyakov, L Wu, S Wu, E Lacy. US Patent, US 2013/0090457 A1 (2013). IMGENEX products cited: TLR3 (IMG-315A).
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Research purposes only. Not for diagnostic or in vivo use.