ULBP-2/5/6 Antibody [Unconjugated]

ULBP-2/5/6 was detected in immersion fixed paraffin-embedded sections of human colon cancer tissue using 10 µg/mL Goat Anti-Human ULBP-2/5/6 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1298) overnight at 4 °C. Before incubation with the primary antibody tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained with the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Rh159 interferes with intracellular transport of NKG2DL.A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (D), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/27580123), licensed under a CC-BY license. Not internally tested by R&D Systems.

Rh159 interferes with intracellular transport of NKG2DL.A) Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. B) Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. C) Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. D-E) Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (D), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/27580123), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, IHC, B/N
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Concentration: LYOPH

ULBP-2/5/6 Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human ULBP-2
  Gly26-Ser217
  Accession # Q9BZM5
• Specificity: Detects human ULBP-2, ULBP-5, and RAET1L/ULBP-6 in Western blots. In direct ELISAs and Western blots, less than 2% cross-reactivity with recombinant human (rh) ULBP-1, rhULBP-3, and rhULBP-4 is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Purity Statement: Antigen Affinity-purified
• Endotoxin Note: <0.10 EU per 1 μg of the antibody by the LAL method.

Applications/Dilutions

• Dilutions:
  • Blockade of Receptor-ligand Interaction
  • Immunohistochemistry 5-15 ug/mL
  • Western Blot 0.1 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.2 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for ULBP-2/5/6 Antibody [Unconjugated]

• ALCAN-alpha
• N2DL2
• NKG2DL2
• RAET1H
• RAET1L
• UL16 binding protein 2
• ULBP2
• ULBP-2/5/6

Background

ULBPs activate multiple signaling pathways in primary NK cells, resulting in the production of cytokines and chemokines. Binding of ULBPs ligands to NKG2D induces calcium mobilization and activation of the JAK2, STAT5, ERK and PI3K kinase/Akt signal transduction pathway. The name ULBP derives from the original identification of three proteins, ULBP-1, -2, and -3, as ligands for the human cytomegalovirus glycoprotein UL16; they were designated UL16 binding proteins (ULBP). The genes for ULBPs reside in a cluster of ten related genes, six of which encode potentially functional glycoproteins. ULBP-2 has also been described under the names RaeT1H (retinoic acid early transcript), NKG2DL2, and ALCAN-alpha. ULBP-5 also known as RaeT1G and ULBP-6 also known as RaeT1L. These proteins are distantly related to MHC class I proteins, but they possess only the alpha 1 and alpha 2 Ig-like domains, and they have no capacity to bind peptide or interact with
beta 2‑microglobulin. Some family members, including ULBP-2, are anchored to the membrane via a GPI-linkage, whereas others have transmembrane domains. Engagement of NKG2D results in the activation of cytolytic activity and/or cytokine production by these effector cells. The ULBPs are expressed on some tumor cells and have been implicated in tumor surveillance. Over aa 26-217, ULBP-2 shares 92% and 95% aa sequence identity with the human ULBP-5 and ULBP-6, respectively.

1. Cosman, D. et al. (2001) Immunity 14:123.
2. Kubin, M. et al. (2001) Eur. J. Immunol. 31:1428.
3. Sutherland, C. et al. (2002) J. Immunol. 168:671.
4. Steinle, A. et al. (2001) Immunogenetics 53:279.
5. Sutherland, C. et al. (2001) Immunol. Rev. 181:185.
6. Pende, D. et al. (2002) Cancer Res. 62:6178.
7. Radosavljevic, M. et al. (2002) Genomics 79:114.
8. NKG2D and its Ligands (2002) www.RnDSystems.com.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for ULBP-2/5/6 Antibody (AF1298)(16)

Publications using AF1298

• Ceri Alan Fielding, Pragati Sabberwal, James C Williamson, Edward J D Greenwood, Thomas W M Crozier, Wioleta Zelek et al. SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies eLife 5/19/2022 [PMID: 35587364]
• J Joung, PC Kirchgatte, A Singh, JH Cho, SP Nety, RC Larson, RK Macrae, R Deasy, YY Tseng, MV Maus, F Zhang CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity Nature Communications, 2022-03-25;13(1):1606. 2022-03-25 [PMID: 35338135] (Western Blot, Human)
• S Jeon, HK Kim, JY Kwon, SH Baek, HS Ri, HJ Choi, HR Cho, YS Lee, JY Kim, J Kim, J Bae, HJ Lee Role of Sevoflurane on Natural Killer Group 2, Member D-Mediated Immune Response in Non-Small-Cell Lung Cancer: An In Vitro Study Med Sci Monit, 2020-11-03;26(0):e926395. 2020-11-03 [PMID: 33139690] (ICC, Human)
• R Okita, A Maeda, K Shimizu, Y Nojima, S Saisho, M Nakata Clinicopathological relevance of tumor expression of NK group 2 member D ligands in resected non-small cell lung cancer Oncotarget, 2019-11-26;10(63):6805-6815. 2019-11-26 [PMID: 31827723] (IHC-P, Human)
• Riki Okita, Ai Maeda, Katsuhiko Shimizu, Yuji Nojima, Shinsuke Saisho, Masao Nakata Effect of platinum‑based chemotherapy on the expression of natural killer group�2 member�D ligands, programmed cell death‑1 ligand�1 and HLA class�I in non‑small cell lung cancer Oncology Reports 6/3/2019 [PMID: 31173242]
• Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus PLoS Pathog, 2016-08-31;12(8):e1005868. 2016-08-31 [PMID: 27580123] (Flow Cytometry, Human)
• Tessa M. Campbell, Brian P. McSharry, Megan Steain, Barry Slobedman, Allison Abendroth Varicella-Zoster Virus and Herpes Simplex Virus 1 Differentially Modulate NKG2D Ligand Expression during Productive Infection Journal of Virology 8/1/2015 [PMID: 25995251]
• Leung W, Vong Q, Lin W, Bouck D, Wendt S, Sullivan E, Li Y, Bari R, Chen T, Leung W PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60. J Immunol, 2015-02-16;194(6):2930-41. 2015-02-16 [PMID: 25687758] (Western Blot, Human)
• Cho , Hanbyoul, Chung , Joon-Yon, Kim , Sunghoon, Braunschweig , Till, Kang , Tae Heun, Kim , Jennie, Chung , Eun Joo, Hewitt , Stephen, Kim , Jae-Hoon MICA/B and ULBP1 NKG2D ligands are independent predictors of good prognosis in cervical cancer. BMC Cancer, 2014-12-15;14(0):957. 2014-12-15 [PMID: 25510288] (IHC-P, Human)
• de Kruijf EM, Sajet A, van Nes JG et al. NKG2D ligand tumor expression and association with clinical outcome in early breast cancer patients: an observational study. BMC Cancer 1/1/2012 [PMID: 22257486]
• Heinemann A, Zhao F, Pechlivanis S, Eberle J, Steinle A, Diederichs S, Schadendorf D, Paschen A Tumor suppressive microRNAs miR-34a/c control cancer cell expression of ULBP2, a stress-induced ligand of the natural killer cell receptor NKG2D. Cancer Res., 2011-11-18;72(2):460-71. 2011-11-18 [PMID: 22102694] (Western Blot, Human)
• Chang YT, Wu CC, Shyr YM, Chen TC, Hwang TL, Yeh TS, Chang KP, Liu HP, Liu YL, Tsai MH, Chang YS, Yu JS Secretome-Based Identification of ULBP2 as a Novel Serum Marker for Pancreatic Cancer Detection. PLoS ONE, 2011-05-20;6(5):e20029. 2011-05-20 [PMID: 21625447] (IHC, Western Blot, Human)
• Fernandez-Messina L, Ashiru O, Aguera-Gonzalez S, Reyburn HT, Vales-Gomez M The human NKG2D ligand ULBP2 can be expressed at the cell surface with or without a GPI anchor and both forms can activate NK cells. J. Cell. Sci., 2011-01-11;124(0):321-7. 2011-01-11 [PMID: 21224393] (ICC, Immunoprecipitation, Human)
• Neil J. Bennett, Omodele Ashiru, Fiona J. E. Morgan, Yin Pang, Georgina Okecha, Rob A. Eagle et al. Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus The Journal of Immunology 7/15/2010 [PMID: 20530255]
• Ohashi M, Eagle RA, Trowsdale J Post-translational modification of the NKG2D ligand RAET1G leads to cell surface expression of a glycosylphosphatidylinositol-linked isoform. J. Biol. Chem., 2010-03-19;285(22):16408-15. 2010-03-19 [PMID: 20304922] (Western Blot, Human)
• Schwinn N, Vokhminova D, Sucker A, Textor S, Striegel S, Moll I, Nausch N, Tuettenberg J, Steinle A, Cerwenka A, Schadendorf D, Paschen A Interferon-gamma down-regulates NKG2D ligand expression and impairs the NKG2D-mediated cytolysis of MHC class I-deficient melanoma by natural killer cells. Int. J. Cancer, 2009-04-01;124(7):1594-604. 2009-04-01 [PMID: 19089914] (Western Blot, Human)

Bioinformatics

• Uniprot:
  • Q9BZM5()