Monoclonal Antibody to TLR9/CD289 (Clone 26C593.2)

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Catalog No	:	IMG-305C
Contents	:	0.1 mg antibody in 0.2 ml buffer (10 mM Tris, 150 mM NaCl and 0.05% sodium azide). Sodium azide is highly toxic.
Isotype	:	Mouse IgG1, Kappa
Clone	:	26C593.2
Purification	:	Protein G Chromatography
Species React	:	Dog, Horse, Human, Mouse, Rat, Rhesus Monkey
Host	:	Mouse

Application
Western blot analysis: 1-3 ug/ml
Flow (Intracellular): 0.1-1 ug/10^6 cells
IHC (frozen): 10-20 ug/ml (see Product citation, Miller et al, 2005).
IHC (paraffin):see Martin-Armas et al. (2005)
IF/ICC: see Greene et. al. (2005) for details
Flow (Cell Surface): see Lee et al. (2006)
Confocal Microscopy: see Greene et. al. (2005)
Storage
Product is light- and temperature-sensitive. Store 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, 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). By using a BLAST search, Hemmi et al., 2000 (4) have identified and subsequently isolated a cDNA coding for TLR9. Gene knockout experiments suggest that TLR9 acts as a receptor for unmethylated CpG dinucleotides in the bacterial DNA (4). Human and mouse TLR9 share an overall amino-acid identity of 75.5%. TLR9 is highly expressed in spleen.

Antigen
This antibody was developed against a KLH-conjugated synthetic peptide corresponding to amino acids 268-284 (CPRHFPQLHPDTFSHLS) of human TLR9.

Genebank Info (Protein)
NP_059138

Intracellular flow analysis of TLR9 in Ramos cells using 0.5 ug of IMG-305C. Green represents isotype control (IMGENEX, 20105); red represents anti-TLR9 antibody. IMGENEX's TLR intracellular flow kit (IMGENEX, 10098K) was used for this test.

Intracellular flow cytometric analysis of TLR9 in human B cells using 1ug/10^6 cells of IMG-305C. Cells were primarily stained with PE-conjugated CD19 antibody (IMGENEX, IMG-6321D). Cells were then fixed and permeabilized and stained with 1ug of either isotype control antibody (IMGENEX, 20105) or IMG-305C. Green represents isotype control; red represents anti-TLR9 antibody.

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. Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, Takeda K, and Akira S. Gene 231: 59-65 (1999).
5. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, and Shizo A. Nature 408: 740-745 (2000).

Product Citations
1. Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by lactamase enzyme fragment complementation. Hyun-Ku Lee, Stefan Dunzendorfer, and Peter S. Tobias. J. Biol. Chem., Dec 2003; 10.1074/jbc.M311564200. Imgenex antibodies cited: TLR9 (IMG-305) [WB, Fig5B (HEK293 cells)].
2. TGF-a Regulates TLR Expression and Function on Epidermal Keratinocytes. Lloyd S. Miller, Ole E. Sørensen, Philip T. Liu, H. Ray Jalian, Deborah Eshtiaghpour, Behnaz E. Behmanesh, Woosin Chung, Timothy D. Starner, Jenny Kim,, Peter A. Sieling, Tomas Ganz and Robert L. Modlin. J. Immunol, 174: 6137-6143 (2005). Imgenex antibodies cited: 1. TLR5 (IMG-664) [IHC-frozen, Fig 1 (normal human skin and psoriasis, primary human organotypic keratinocyte culture)]. 2. TLR9 (IMG-305A) [IHC-frozen, Fig 1 (normal human skin and psoriasis, primary human organotypic keratinocyte culture)].
3. EBV Latent Membrane Protein 2A Induces Autoreactive B Cell Activation and TLR Hypersensitivity. Hongsheng Wang, Matilda W. Nicholas, Kara L. Conway, Pradip Sen, Ramiro Diz, Roland M. Tisch, and Stephen H. Clarke. J. Immunol., 177: 2793-2802 (2006). (Flow)
4. CpG DNA activation and plasma cell differentiation of CD27^- naive human B cells. Jennifer Huggins, Tina Pellegrin, Raymond E. Felgar, Chungwen Wei, Miguel Brown, Bo Zheng, Eric C.B. Milner, Steven H. Bernstein, Ignacio Sanz, and Martin S. Zand. Blood, Oct 2006; 10.1182/blood-2006-03-008441. (Flow)
5. Members of the Toll-like receptor family of innate immunity pattern-recognition receptors are abundant in the male rat reproductive tract. MA Palladino, TA Johnson, R Gupta, JL Chapman, P Ojho. Biol Reproduction 76:958-964 (2007). Imgenex anitbodies cited: The following antibodies were used for WB in Fig 2 in rat testis, epididymis, and vas deferens: TLR1 (IMG-5012), TLR2 (IMG-545), TLR5 (IMG-580), TLR6 (IMG-527), TLR7 (IMG-581), TLR9 (IMG-305A), and TLR12 (IMG-5034).

MYD88 (IMG-178) and Trif (IMG-661) were used for WB in Fig 3 in rat testis, epididymis, vas deferens, and mouse Raw 264 cells.
6. 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).
7. Activation of anti-hepatitis C virus responses via Toll-like receptor 7. Lee J, CCN Wu, KJ Lee T-H Chuang, K Katakura, Y-T Liu, M Chan, R Tawatao, M Chung, C Shen, HB Cottam, MMC Lai, E Raz and DA Carson. PNAS 103:1828-1833 (2006). Imgenex antibodies cited in this study:  Flow (Intracellular) and Flow (Cell Surface) in human Huh-7 hepatoma cells, Fig. 1A [TLR7 (IMG-665A], [TLR8 (IMG-321C)], and [TLR9 (IMG-305D)]; IHC (paraffin) on Huh-7 cells, normal fibroblasts, HCV-infected liver, and liver carcinoma, Fig 1C-E (TLR7, IMG-665A). The specificity of the TLR7 antibody (IMG-665A) was validated using TLR7 293 transfected cells in Flow (Intracellular) (Supplemental Data, Fig. 5).
8. Surface expression of Toll-like receptor 9 is upregulated on intestinal epithelial cells in response to pathogenic bacterial DNA. Ewaschuk JB, JL Backer, TA Churchill, F Obermeier, DO Krause, and KL Masden. Infection and Immunity 75:2572-2579 (2007). Imgenex antibody cited: IMG-305A in human HT-29 colon carcinoma cells Flow (Intracellular) Fig 4, IF (Fig 5), WB (Fig 6) and in mouse colon tissue (from normal and germ-free mice) for WB (Fig 7A, B) and IHC (paraffin) (Fig 7C).
9. Toll-like receptor 9 expression in murine and human adrenal glands and possible implications during inflammation. Tran N, A Koch, R Berkels, O Boehm, PA Zacharowski, G Baumgarten, P Knuefermann, M Schott, W Kanczkowski, SR Bornstein, SL LIghtman, and K Zacharowski. J Clin Endocrin Metab. Doi:10.1210/jc.2006-2697 In press (2007). Imgenex antibodies cited (mouse spleen and adrenal tisse, mouse RAW264.7 macrophage cell line, human adrenal tissue: TLR9 [(IMG-305A) IHC (paraffin): Figs 1C-D, F and 6; IF/ICC: Figs 1E and 5]; TLR9 [(IMG-431) WB: Figs 1B, 3C, and 5G]. The TLR9 IMG-431 antibody was validated by WB using adrenal gland tissue from TLR9^-/- and wt mice (Fig 1B).
10. Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in Rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing Rhesus monkey plasmacytoid dendritic cells. Abel K, Y Wang, L Fritts, E Sanchez, E Chung, P Fitzgerald-Bocarsly, AM Krieg, and CJ Miller. Clinical and Diagnostic Laboratory Immunology 12:606-621 (2005). Imgenex antibodies cited: IMG-305C [Flow (Intracellular), Fig. 5] on Rhesus monkey spleen cell suspensions and PBMC.
11. 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.]
12. Deviation from major codons in the Toll-like receptor genes is associated with low Toll-like receptor expression. Fei Zhong, Weiping Cao, Edmund Chan, Puei Nam Tay, Florence Feby Cahya, Haifeng Zhang and Jinhua Lu. Immunology, 114 (1) Page 83 (2005). Imgenex antibodies cited: 1. TLR2 (IMG-416) 2. TLR9 (IMG-305) [Flow, Fig.1 (293T cells and monocytes)].
13. TLR-Induced Inflammation in Cystic Fibrosis and Non-Cystic Fibrosis Airway Epithelial Cells. Catherine M. Greene, Tomás P. Carroll, Stephen G. J. Smith, Clifford C. Taggart, James Devaney, Siobhan Griffin, Shane J. O’Neill, and Noel G. McElvaney. J. Immunol., 174: 1638-1646 (2005). Imgenex antibodies cited: 1. TLR9 (IMG-305) [Confocal Microscopy, Fig.1A and 1B (CFTE29o- and 16HBE14o- cells)].
14. Maintenance of colonic homeostasis by distinctive apical TLR9 signaling in intestinal epithelial cells. Lee J., J-H Mo, K. Katakura, I. Alkalay, A. N. Rucker, Y-T. Liu, H-K. Lee, C. Shen, G. Cojocaru, S. Shenouda, M. Kagnoff, L. Eckmann, Y. Ben-Neriah, and E. Raz., Nature Cell Biol., 8,doi:10.1038/ncb1500 (2006). Imgenex antibodies cited: 1. TLR9 (IMG-305) [Confocal Microscopy, Fig.2 (intestinal epithelial cells)]. [Flow (cell surface), Fig.S1 (293-HEK TLR9 transfected cells) and Fig.S2 (Caco-2 intestinal epithelial cells)], TLR9 mAb was transfected validated by flow (cell surface) in Fig. S1. [WB and IP, Fig S1 (293-HEK TLR9 transfected cells and Caco-2 intestinal epithelial cells), TLR9 mAb was transfected validated by IP/WB.

15. Role of pathogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemic lupus erythematosus. Nakano S, S Morimoto, J Suzuki, K Nozawa, H Amano, Y Tokano, Y Takasaki. Rheumatology 47: 145-149 (2008). Imgenex antibody cited: IMG-305C (TLR9-FITC) for Flow (Intracellular), human peripheral blood B cells from normal and SLE patients, Figs 1C, 2, 3.
16. Induction of pro-inflammatory programs in enteroendocrine cells by the Toll-like receptor agonists flagellin and bacterial LPS. Selleri S, Palazzo M, Deola S, Wang E, Balsari A, Marincola F, Rumio C. International Immunology doi:10.1093/intimm/dxn055 (2008). TLR9-FITC (IMG-305C): Flow (intracellular), Human colon neuroendocrine LCC-18 cell line: Fig. 4d.
17. Expression of TLR9 within human glioblastoma. Meng Y, M Kujas, Y Marie, S Paris, J Thillet, J Delattre, A Carpentier. Journal of Neuro-Oncology: 10.1007/s11060-008-9536-2 (2008). IMG-305A: human glioma cell lines SNB19, HTB13 and U87, IHC(parrafin): Fig. 2a; IF: Fig. 2c,f.
18. TLR9 is expressed in idiopathic interstitial pneumonia and its activation promotes in vitro myofibroblast differentiation. Meneghin A, E Choi, H Evanoff, S Kunkel, F martinez, K Flaherty, G Toews, C Hogaboam. Histochemisty and Cell Biology: 10.1007/s00418-0080466-z (2008). Imgenex products cited (human lung fibroblast cell lines):
1. IMG-305A (TLR9): IHC (paraffin), Fig. 2a,b,c,d,e,f; 3b,d; 4a,b,c,d,e,rf,g,h; 5b,d,f; 6b,d,f,h.
2. IMG-305B (TLR9 biotin): IF, Fig. 8a,b,c,d.
19.

STAT3 and STAT5-dependent pathways competitively regulate the pan-differentiation of DC34^pos 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 cited [Flow (intracellular), mouse bone marrow cells, Supplementary Fig. S4]:
1. TLR3 FITC (IMG-315C)
2. TLR4 FITC (IMG-5031C)
3. TLR7 (IMG-665A)
4. TLR8 FITC (IMG-321C)
5. TLR9 FITC (IMG-305C)

20. CD300a/c regulate type 1 intergeron and TNF-α secretion by human plasmacytoid dendritic cells stimulated with TLR7 and TLR9 ligands. Ju X, M Zenke, D Hart, G Clark. Blood 112: 1184-1194 (2008). IMG-305D: Flow (cell surface), human PBMC cells, Fig 1B.
21.

Molecular cloning and characterization of equine Toll-like receptor 9. Zhang YW, EG Davis, F Blecha, MJ Wilkerson. Veterinary Immunology and Immunopathology 124: 208-219 (2008)..

Imgenex Products cited for Flow (Cell Surface) and Flow (Intracellular):.

IMG-305C (TLR9-FITC) and IMG-305D (TLR9-PE): Fig 2B (HEK293 and equine TLR9 transfected cells), Fig 4 (equine leukocytes), Fig 5 (equine PHA-stimulated PBMC), Fig 6 (equine leukocyte subsets). .

Notes: TLR9 expression was found to be predominantly intracellular. HEK293 cells express endogenous TLR9. TLR9 expression increased in equine TLR9 transiently transfected HEK293 cells. TLR9 expression increased in PBMC cells following PHA stimulation.

22.

Antibodies specific for human or murine Toll-like receptors detect canine leukocytes by flow cytometry. Burgener IA and TW Jungi. Veterinary Immunology and Immunopathology124:184-191 (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

23. Inhibitors of TLR8 reduce TNF production from human rheumatoid synovial membrane cultures. Sacre S, A Lo, B Gregory, R Simmonds, L Williams, M Feldmann, F Brennan, B Foxwell. J Immunol 181: 8002-8009 (2008). Imgenex antibodies cited [Flow (intracellular), human synovial membrane cells, Fig. 4B]:
1. TLR3-FITC (IMG-315C)
2. TLR8-FITC (IMG-321C)
3. TLR9-FITC (IMG-305C).
24. Role for plasmacytoid dendritic cells in the immune control of recurrent human herpes simplex virus infection. Donaghy H, L Bosnjak, A Harman, V Marsden, S Tyring, T Meng, A Cunningham. Journal of Virology 83: 1952-1961 (2008). IMG-305C: Human plasmacytoid dendritic cells, Flow(cell surface), Fig. 3A(left); Flow (intracellular), Fig. 3A (right).
25.

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).

26. Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway. Nakamura K, A Miyazato, G Xiao, M Hatta, K Inden, T Aoyagi, K Shiratori, K Takeda, S Akira, S Saijo, Y Iwakura, Y Adachi, N Ohno, K Suzuki, J Fujita, M Kakau, and K Kawakami. J Immunol 180:4067-4074 (2008). Imgenex antibody used: TLR9-FITC (IMG-305C) for IF/ICC (intracellular confocal microscopy) in the following figures:

1. Fig 8a: primary mouse bone marrow-derived myeloid dendritic cells (BM-DC) treated with Cn-DNA and CpG-Rhodamine and analyzed over a time course

2. Fig 8b: BM-DC and RAW264.1 cells treated with Cn-DNA

Note: intracellular redistribution of TLR9 was observed in treated cells

27. The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Koichi Tabeta, Kasper Hoebe, Edith M Janssen, Xin Du, Philippe Georgel, Karine Crozat, Suzanne Mudd, Navjiwan Mann, Sosathya Sovath, Jason Goode, Louis Shamel, Anat A, et al. Nature Immunology 7, 156-164 (2006). Imgenex antibodies cited: 1. TLR9 (IMG-305) [IF/ICC, Fig.5g-i (primary mouse macrophages)]. The specificity of the TLR9 antibody is demonstrated using primary mouse macrophages from wildtype (5g) and TLR9 knockout (5i) mice.
28. Nasolacrimal duct closure modulates ocular mucosal and systemic CD4+ T cell responses induced following topical ocular or intranasal immunization. Chentoufi A, G Dasgupta, A Nesburn, I Bettahi, N Binder, Z Choudhury, W Chamberlain, S Wechsler, L Benmohamed. Clin Vacc Immunol 17: 342-353 (2010). IMGENEX products cited:
1. TLR9 (IMG-305A): WB, Fig 7 rabbit CD11b+ cells.
2. TLR9 PE (IMG-305D): Flow (Intracellular), Fig 7A stained CD11b+ cells (isolated from eye conjunctiva).
IHC (frozen), Fig 7D CD11b+ (isolated from eye conjuctiva and spleen).
29. Protein kinase D1 is essential for the proinflammatory response induced by hypersensitivity phemonitis-causing thermophilic actinomycetes saccharopolyspora rectivirgula. Kim Y, J Park, D Brank, E Firzpatrick, A Yi. J Immunol 184: 3145-3156 (2010). IMGENEX product cited: TLR9 (IMG-305A) WB, Fig 2, lung cells from MyD88 deficient mice.
30. Expression of Toll-like receptor 9 in renal podocytes in childhood-onset active and inactive lupus nephritis. Machida H, S Ito, T Hirose, F Takeshita, H Oshiro, T Nakamura, M Mori, Y Inayama, K Yan, N Kobayashi, S Yokota. Nephrol Dial Transplant doi: 10.1093/ndt/gfq058 (2010) IMGENEX product cited: TLR9 (IMG-305A) IHC (paraffin), Fig 2A, human normal kidney control.  Fig 2B, Fig 2C, active lupus kidney.
31. Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response. Panda A, F Qian, S Mohanty, D Duin, F Newman, L Zhang, S Chen, V Towle, R Belshe, E Fikrig, H Allore, R Montgomery, A Shaw. J. Immunol 184: 2518-2527 (2010). IMGENEX products cited:
1. TLR9 FITC (IMG-305C) Flow (intracellular) Fig 2, Fig 6, human myeloid dendritic and human myeloid dendritic cells.
2. TLR8 Biotin (IMG-321B) Flow (intracellular) Fig 2, Fig 6, human myeloid dendritic and human myeloid dendritic cells.
32. Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of Toll-like receptor 9. Kindrachuk J, E Potter, R Brownlie, A Ficzycz, P Griebel, N Mookherjee, G Mutwiri, L Babiuk, S Napper. J Bio Chem 282: 13944-13953 (2007). IMGENEX product cited: TLR9 (IMG-305A)
1. WB, Fig 1: TLR9 stably transfected bovine kidney epithelial cells, these cells secrete recombinant TLR9 (ligand binding domain) into the media. The data shows the molecular weight of TLR9 was reduced following deglycosylation.
2. EMSA, Fig 2: TLR9 formed monomeric and dimeric nucleoprotein complexes with the plasmid CMV. Protein component of shifted band was excised from the gel then subjected to western blot analysis (Data not shown).
3. EMSA, Fig 3. TLR9 plasmid binding is enhanced at acidic pH values. Fig 6, TLR9 is highly sequence-specific in plasmid binding.
33. Expression and characterization of ligand binding by the ectodomain of Toll-like receptor 9. E. A. Potter, (2007) Dept. of Biochemistry University of Saskatchewan. IMGENEX product cited: TLR9 (IMG-305A)
1. WB, Fig 4.4, TLR9 stably transfected Madin-Darby bovine kidney epithelial cells, these cells secrete recombinant TLR9 (ligand binding domain) into the media. The data shows the molecular wieght of TLR9 was reduced by deglycosylation.
2. EMSA, Fig 4.5: TLR9 formed monomeric and dimeric nucleoprotein complexes with the plasmid CMV. Protein component of shifted band was excised from the gel then subjected to western blot analysis, Fig 4.6
34. Chicken TLR21 is an innate CpG DNA receptor distinct from Mammalian TLR9. Keestra M, M Zoete, L Bouwman, J Putten. J Immunol 185: 460-467 (2010). Confocal microscopy: HEK293 cells transfacted with hTLR9, Fig 6A.

Note: Monoclonal TLR9 antibody was transfected validated in TLR9 transfected HEK293 cells by confocal microscopy, Fig 6A.
35. 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).
36. 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).

37. Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis related arthritis subtype of juvenile idiopathic arthritis. Rheumatology 50:481-488 (2011). IMGENEX product cited: TLR9/CD289 PE (IMG-305D). Flow (Intracellular): Human PBMCs and SF mononuclear (SFMCs) cells, Fig 1 & 2.
38. EBV Lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection. Gent M, B Griffin, E Berkhoff, D Leeuwen, I Boer, M Buisson, F Hartgers, W Burmeister, E Wiertz, M Ressing. J Immunol 186:0-0 (2010). IMGENEX product cited: TLR9/CD289 (IMG-305A).
WB: AK31 and AKBM cell postnuclear lysates infected with Epstein-Barr virus, Fig 2B.
WB: TLR9 transfected 293T cells, Fig 4D.

Note: The TLR9 antibody (IMG-305A) was transfected validated by WB in 293T cells, Fig 4D.

39. Identification of two subpopulations of purified human blood B cells, CD27- CD23+ and CD27high CD80+, that strongly express cell surface Toll-like receptor 9 and secrete high levels of interleukin-6. Cognasse F, H Cognasse, S Lafarge, P Chavarin, B Pozzetto, Y Richard, O Garraud. J Immunol 125:430-437 (2007). IMGENEX products cited: Human intestine tissue lysate (40143) and TLR9/CD289 (IMG-305A). WB: Human intestine lysate was probed with TLR9 antibody, Fig 1.
40. Localization of toll-like receptors on epididymal epithelial cells and spermatozoa. Palladino M, M Savarese, J Chapman, M Dughi, D Plaska. Am J Reprod Immunol 60:541-555 (2008). IMGENEX products cited: TLR1 (IMG-5012), TLR2 (IMG-545), TLR5 (IMG-580), TLR6 (IMG-527), TLR9 (IMG-305A), and TLR11 (IMG-5034) antibodies; Ramos Cell (40175) and Mouse Heart Tissue (40102) Lysates.
1. IHC (paraffin): Rat spleen stained with TLR 1 & 2, Fig 1.
2. IHC (paraffin): Staining rat cauda epididymidis with TLR5 and TLR6, Fig 4.
3. IHC (paraffin): Staining rat initial segment and cauda epididymidis with TLR9, Fig 5.
4. IHC (paraffin): Staining rat cauda epididymidis with TLR 11, Fig 6.
5. WB: Ramos and Mouse Heart Lysate, Fig 7.
41. Immunolocalization of pulmonary intravascular macrophages, TLR4, TLR9 and IL-8 in normal and pastuerella mutocida-infected lungs of water buffalo (Balbalus bubalis). Sethi RS, RS Bra O Singh, B Singh. J Comp Pathol 144:135-144 (2011). IMGENEX antibody cited TLR9 (IMG-305A) for IHC (P): Fig 5a-f (water buffalo lung lungs). Inflamed lung showed increased TLR9 expression in the alveolar septa, airway epithelium, vascular endothelium, and blood cells as compared to normal lung.
42. Human papillomavirus deregulates the response of a cellular network comprising of chemotactic and proinflammatory genes. Karim R, C Meyers, C Backendorf, K Ludigs, R Offringa, G Melief, S Burg, J Boer. PLoS ONE 6:e17848 (2011). IMGENEX product cited: TLR9/CD289 (IMG-305A). IHC (paraffin): Human foreskin, cervical and vaginal keratinocyte tissue, Fig 3.
43. Toll like receptor expression in normal ovary and ovarian tumors. Zhou M, M Mancini, H Funk, N Husseinzadeh, T Mounajjed, A Drew. Cancer Immunology 58:1375-1385 (2009). IMGENEX product cited: TLR7 (IMG-665A) & TLR9/CD289 (IMG-305A). IHC (paraffin): Human normal ovary, serous papillary, and clear cell ovarian carcinomas, Fig 1.
44. NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis. Souza-Fonseca-Guimaraes F, M Parlato, C Fitting, J-M Cavaillon, M Adib-Conguy. J Immunol 188:5850-5858 (2012). Imgenex antibody cited in this study for Flow (Intracellular): TLR9-AF488 (IMG-305AF488):
Fig 1A &B (mouse peritoneal macrophages).
Fig 2C (mouse NK cells isolated from spleen).
45.

The circadian clock controls Toll-like receptor 9-mediated innate and adaptive immunity. Silver AD, A Arjona, WE Walker, E Fikrig. Immunity 36:251-261 (2012).

1832-1843 (2008). Imgenex antibodies cited (mouse spleen, splenic macrophages, splenic B cells, splenic CD11b+ cells, splenic CD19+ spleen cells):
1. TLR9 (IMG-305A): WB, Figs 2, S2

2. TLR9-FITC (IMG-305C): Flow (Intracellular)

46.

Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity. Zuo D, X Yu, C Guo, H Yi, X Chen, DH Conrad, TL Guo, Z Chen, PB Fisher, JR Subjeck, X-Y Wang. FASEB J 26:1493-1505 (2012). Imgenex antibody cited: TLR9 IMG-305A (Raw cells and mouse bone marrow derived macrophages): Fig 4 (IF, IP, IP/WB, WB).

Note: The TLR9 antibody co-IP'd His-Grp170. IF studies with the TLR9 antibody also showed that TLR9 and and internalized Grp170 colocalized in endosomes.

47. CLINICAL AND HUMAN IMMUNOLOGY: Identification of Lineage Relationships and Novel Markers of Blood and Skin Human Dendritic Cells. Harman A., C. Bye, N. Nasr, K.Sandgren, M. Kim, S. Mercier, RA Botting, SR Lewin, AL Cunningham, PU Cameron. J Immunol. 190:66-79 doi:10.4049/jimmunol.1200779 ( 2013)

IMGENEX products cited:

All testing was done on dendritic cells.
IMG 664A Unconjugated TLR5 mAb (19D759.2) Flow Cell Surface and Intracellular
IMG 304B biotinylated TLR6 mAb (86B1153.2) Flow Cell Surface and Intracellular
unconjugated rabbit polyclonal TLR7 Flow Intracellular
IMG 321C TLR8-FITC (44C143) Flow Intracellular
IMG 305C TLR9-FITC (26C593.2) Flow Intracellular48. Spontaneous production of immunoglobulin m in human epithelial cancer cells. Hu F, L Zhang, J Zheng, L Zhao, J Huang, W Shao, Q Liao, T Ma, L Geng, CC Yin and X Qiu. PLoS doi:10.1371/journal.pone.0051423 (2012).IMGENEX products cited: TLR 9 (IMG-305A).49. Expression of toll-like receptor 9 in lungs of pigs, dogs and cattle. Schneberger D, D Lewis, S Caldwell & B Singh. International J Experimental Pathol. DOI: 10.1111/j.1365-2613.2010.00742.x (2010). IMGENEX products cited: TLR9 (305A).50. Distinct autoantibody profiles in systemic lupus erythematosus patients are selectively associated with TLR7 and TLR9 upregulation. Chauhan SK, VV Singh, R Rai, M Rai, G Rai. J Clin Immunol DOI 10.1007/s10875-013-9887-0 (2013). IMGENEX antibodies cited for PBLs (WB) or B-cells (Flow intracellular) from SLE patients or controls:
1. TLR7 mAb clone IMG4G6 (IMG-6675A): WB (Figs 2a, 2c)
2. TLR7-PE mAb, clone IMG4G6 (IMG-6675D): Flow intracellular (Figs 3a, 3c)
3. TLR9 mAb, clone 26C593.2 (IMG-305A): WB (Fig 2b, 2d)
4. TLR9-FITC mAb, clone 26C593.2 (IMG-305C): Flow intracellular (Figs 3b, 3d)
5. GAPDH pAb (IMG-5143A): WB (Fig 2)
6. Mouse IgG1-PE isotype control, clone MOPC-31C (20106): Flow intracellular (Figs 3a, 3c)
7. Mouse IgG1-FITC isotype control, clone MOPC-31C (20104): Flow intracellular (Figs 3b, 3d)

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