Anti ulbp3

Manufactured by R&D Systems

Sourced in United States

Anti-ULBP3 is a recombinant protein that specifically binds to the ULBP3 molecule. ULBP3 is a ligand for the NKG2D receptor, which is expressed on the surface of natural killer cells, CD8+ T cells, and other immune cells. Anti-ULBP3 can be used in research applications to study the interactions between ULBP3 and NKG2D.

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Lab products found in correlation

Anti ulbp1, by R&D Systems (4 mentions) Anti ulbp2, by R&D Systems (2 mentions) Anti micb clone 236511, by R&D Systems (2 mentions) Anti mica, by R&D Systems (2 mentions) Anti ulbp 2 5 6, by R&D Systems (2 mentions) Anti cd56, by BD (1 mentions) Anti cd3, by BioLegend (1 mentions) Anti nkg2d antibody, by R&D Systems (1 mentions) Anti cd107a lamp 1, by BioLegend (1 mentions) Anti ceacam 1 clone asl 32, by BioLegend (1 mentions) Mouse igg1 clone mopc 21, by BioLegend (1 mentions) Igg2a clone mopc 173, by BioLegend (1 mentions) Anti mica clone 159227, by R&D Systems (1 mentions) Anti pd l1 mab, by Thermo Fisher Scientific (1 mentions) Fitc mouse igg1, by BioLegend (1 mentions) Pomalidomide, by Merck Group (1 mentions) Anti cd107a fitc h4a3, by BD (1 mentions) Anti cd138 fitc m15, by BD (1 mentions) Anti cd38 apc hit2, by BD (1 mentions) Lenalidomide, by Abcam (1 mentions)

Spelling variants of this product

ULBP3 (7 citations) Anti-ULBP3-PE (5 citations)

7 protocols using anti ulbp3

Quantification of ULBP Proteins by ELISA and Flow Cytometry

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Anti-ULBP1, anti-ULBP2 and anti-ULBP3 antibodies were purchased from R&D systems (catalog numbers: MAB1380, 1248 and 1517 respectively) and used both for flow cytometry and for ELISA assays. Anti-ULBP1 antibody, (catalog number Sc33564, Santa Cruz Biotechnology) was used for western blotting of ULBP1. The W6/32 mAb was used for MHC-I staining. Anti-NKG2D antibody was purchased from R&D Systems (MAB139). The anti-CD99 (12E7) was used as an isotype control. Anti-CD107a (LAMP-1) was purchased from BioLegend (catalog number 328620). Anti-CD56 (Becton Dickinson) and anti-CD3 (BioLegend) antibodies were used to determine NK purity. Anti-VP2/3 and agnoprotein antibodies were produced in house as well as the rabbit polyclonal antibodies against VP1. Anti-T-Ag antibody was purchased from Abcam (Pab416). The commercial recombinant ULBP-1 Fc chimeric protein (R&D systems, catalog number 1380-UL) was used for the generation of ULBP1 standard curve.
CD16-Ig, NKp30-Ig and NKp46-Ig and NKG2D-Ig fusion proteins were generated in the human embryonic kidney 293T cells and were purified on a protein G column as described [38 (link)]. The fusion proteins used in this work were regularly assayed by SDS-PAGE protein gels, to ensure that the proteins were not degraded. Protein purity of all Ig fusion proteins used in this study was approximately 100%.

Bauman Y., Drayman N., Ben-Nun-Shaul O., Vitenstein A., Yamin R., Ophir Y., Oppenheim A, & Mandelboim O. (2016). Downregulation of the stress-induced ligand ULBP1 following SV40 infection confers viral evasion from NK cell cytotoxicity. Oncotarget, 7(13), 15369-15381.

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Flow Cytometry Immunophenotyping of Melanoma Cells

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Melanoma 624 wt or knockout cells were plated at equal densities and incubated overnight. Resuspended cells were incubated on ice for 1 h with the primary antibody at a concentration of 0.2 μg/well in FACS buffer (1× PBS, 0.5% bovine serum albumin, 0.05% NaN₃). The cells were then incubated for 30 min on ice with anti-mouse AlexaFluor 647 secondary antibody (Jackson ImmunoResearch). The following primary antibodies were used: anti-MICA (clone 159227, R&D Systems), anti-MICB (clone 236511, R&D Systems), anti-ULBP1 (clone 170818, R&D Systems), anti-ULBP2/5/6 (clone 165903, R&D Systems), anti-ULBP3 (clone 166514, R&D Systems), anti-B7H6 (clone 875001, R&D systems), anti-PVR (in-house developed), anti-HLA1 (W6/32), anti-Beta-2 microglobulin (β2M, clone 2M2, Biolegend), anti-Ceacam-1 (clone ASL-32, Biolegend), anti-Nectin-2 (clone TX31, Biolegend). Mouse IgG1 (clone MOPC-21, Biolegend), IgG2a (clone MOPC-173, Biolegend), and IgG2b (clone MPC-11, Biolegend) were used as an isotype control.

Obiedat A., Charpak-Amikam Y., Tai-Schmiedel J., Seidel E., Mahameed M., Avril T., Stern-Ginossar N., Springuel L., Bolsée J., Gilham D.E., Dipta P., Shmuel M., Chevet E., Mandelboim O, & Tirosh B. (2019). The integrated stress response promotes B7H6 expression. Journal of Molecular Medicine (Berlin, Germany), 98(1), 135-148.

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Immune Checkpoint Surface Expression

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Surface staining was performed using the following anti-mouse monoclonal antibodies (mAbs) or antibody controls: PE-conjugated IgG isotype and anti-MICA/B, anti-MICA, anti-MICB, anti-ULBP1, anti-ULBP2, anti-ULBP3, anti-HLA-ABC mAb (R&D Systems, Minneapolis, MN, USA), anti-PD-L1 mAb (eBioscience, San Diego, CA, USA), Percp-cy5.5-conjugated IgG isotype and anti-CD95 mAb . Cells were collected with

Guan Y., Li W., Hou Z., Han Q., Lan P., Zhang J., Tian Z, & Zhang C. (2016). HBV suppresses expression of MICA/B on hepatoma cells through up-regulation of transcription factors GATA2 and GATA3 to escape from NK cell surveillance. Oncotarget, 7(35), 56107-56119.

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Immunophenotyping of Tumor Cells and Natural Killer Cells

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Lenalidomide was purchased from (BioVision Inc.), Pomalidomide and Phthalimide were purchased from (Sigma-Aldrich). These drugs were dissolved in dimethylsulphoxide (DMSO) and stored at −20°C until use. The final concentration of DMSO in all experiments was < 0.1%.
The following monoclonal antibodies (mAbs) were used for immunostaining or as blocking Abs: anti-MICA (MAB159227), anti-MICB (MAB236511), anti-ULBP-1 (MAB170818), anti-ULBP-2/5/6 (MAB165903), anti-ULBP-3 (MAB166510) and anti-NKG2D (MAB149810) from (R&D System), anti-PVR/CD155 (SKII.4) kindly provided by Prof. M. Colonna (Washington University, St Louis, MO), anti-CD56 (C218) mAb was provided by Dr. A. Moretta (University of Genoa, Genoa, Italy), anti-DNAM-1 (DX11) from (Serotec), APC Goat anti-mouse IgG (Poly4053), anti-CD3/APC (HIT3a), anti-CD56/PE (HCD56), mouse IgG1/FITC, /PE or /APC isotype control (MOPC-21) were purchased from (BioLegend). Anti-CD107a/FITC (H4A3), anti-CD138-FITC (M15) and anti-CD38-APC (HIT2) were purchased from (BD Biosciences).

Fionda C., Abruzzese M.P., Zingoni A., Cecere F., Vulpis E., Peruzzi G., Soriani A., Molfetta R., Paolini R., Ricciardi M.R., Petrucci M.T., Santoni A, & Cippitelli M. (2015). The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma. Oncotarget, 6(27), 23609-23630.

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Evaluation of Tumor-derived Soluble NKG2DL Inhibition

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T cells were resuspended in complete medium with a density of 1×10⁶ cells/ml and were cultured with anti-CD28 (2 µg/ml) and IL-2 (10 U/ml) in a 24-well plate pre-coated with 5 µg/ml anti-CD3. Subsequently, T cells were co-cultured with or without 100 µl serial diluted tumor sup for 24 h. For neutralizing sNKG2DL, single and combined soluble NKG2DL neutralizing antibodies (1, 2, or 5 µg/ml anti-MICA/B, ULBP1, ULBP2, and/or ULBP3) were added to T cells and to the tumor cell supernatant co-culture system. Then, these T cells were harvested and prepared for flow cytometric analysis. Neutralized antibodies (anti-MICA/B, anti-ULBP1, anti-ULBP2, and anti-ULBP3) were purchased from R&D Systems, Inc. (cat. nos. MAB13001, MAB1380, MAB1298 and MAB1517, respectively).

Zhang Y., Luo F, & Dong K. (2023). Soluble NKG2D ligands impair CD8+ T cell antitumor function dependent of NKG2D downregulation in neuroblastoma. Oncology Letters, 26(1), 297.

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Comprehensive Antibody Panel for Immunoanalysis

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The following primary antibodies were used for flow cytometry: anti-MICA (clone 159227; R&D Systems), anti-MICB (clone 236511, R&D Systems), anti-ULBP1 (clone 170818; R&D Systems), anti-ULBP2/5/6 (clone 165903; R&D Systems) and anti-ULBP3 (clone 166514, R&D Systems).
The following primary antibodies were used for immunofluorescence: anti-PDI (ab3672, Abcam), anti-FLAG tag (Clone L5, Biolegend) and anti-MICA (clone 159227, R&D Systems).
The following primary antibodies were used for western blotting: anti-MICA (Clone EPR6568, Abcam), anti-FLAG tag (Clone L5, Biolegend), anti-GAPDH (clone 6C5, Santa Cruz) and anti-vinculin (clone EPR8185, Abcam).
The following secondary antibodies were used: anti-mouse AlexaFluor 647, anti-mouse PE, anti-mouse biotin, anti-rabbit biotin, anti-rat biotin, anti-rabbit Cy3, anti-rat 488, streptavidin-AlexaFluor 647, streptavidin-horseradish peroxidase (HRP), anti-mouse-HRP, anti-rat-HRP and anti-rabbit-HRP, all purchased from Jackson Laboratories.

Seidel E., Dassa L., Schuler C., Oiknine-Djian E., Wolf D.G., Le-Trilling V.T, & Mandelboim O. (2021). The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing. PLoS Pathogens, 17(5), e1008807.

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Multiparameter Flow Cytometry of Immune Cells

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Cells were washed with fluorescence-associated cell sorting (FACS) buffer (BD Bioscience, 554656) and blocked with Fc blocker (BD Bioscience, 564220) for 30 min at room temperature. Cells were stained with fluorescence-conjugated primary antibodies for 30 min on ice, washed twice with FACS buffer, and fixed with 2% PFA for 15 min. For non-conjugated primary antibodies, the cells were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies for 60 min at room temperature. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). Primary antibodies used were as follows: anti-CD107a (BD Biosciences, 560664), anti-IFNγ (BioLegend, 554701, San Diego, CA, USA), anti-NKG2A (BioLegend, 375103), anti-CD96 (BioLegend, 562379), anti-NKG2D (BD Biosciences, 557940), anti-CD16 (Invitrogen, MHCD1604), anti-ULBP-1 (R&D Systems, MAB1380), anti-ULBP-2/5/6 (R&D Systems, MAB1289), anti-ULBP-3 (R&D Systems, MAB1517), anti-MIC-A/B (Invitrogen, 12-5788-42), anti-HLA-ABC (BD Biosciences, 557349), anti-HAL-E (Invitrogen, 12-9953-41), anti-HLA-G (Invitrogen, 12-9957-41), and CXCR6 (BioLegend, 151103). For CD107a and IFNγ staining, cells were treated with brefeldin A (BioLegend, 420601) for 5 h before incubation with the primary antibody.

Jung H.Y., Lee D.K., Lee M., Choi S.H., Park J.D., Ko E.S., Lee J., Park K.S, & Jung H.Y. (2023). ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer. Oncoimmunology, 12(1), 2190671.

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