CLEC14A Antibody [Unconjugated]

HUVEC human umbilical vein endothelial cells were stained with Sheep Anti-Human CLEC14A Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4968, filled histogram) or isotype control antibody (Catalog # 5-001-A, open histogram), followed by Phycoerythrin-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0126).

CLEC14A was detected in immersion fixed HUVEC human umbilical vein endothelial cells using Sheep Anti-Human CLEC14A Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4968) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces and cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

CLEC14A was detected in formalin fixed paraffin-embedded sections of human breast cancer tissue using Sheep Anti-Human CLEC14A Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4968) at 1.7 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Universal (Catalog # CTS015). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to endothelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

MMRN2 binds to CLEC14A in the C-type lectin domain. (a) CLEC14A-ECD-Fc pull-downs were pre-incubated with either PBS, mouse control IgG, or CLEC14A monoclonal antibodies C1–5 and used to pull down MMRN2FL from HEK293T-transfected lysates. C1, C4 and C5 blocked MMRN2 enrichment, whereas C2 and C3 did not. (b) Flow cytometry analysis of CLEC14A-ECD-Fc binding to HUVEC surface. CLEC14A-ECD-Fc was pre-incubated with the same blocking conditions as in pull-downs. C1, C4 and C5 significantly blocked CLEC14A-ECD-Fc binding to HUVEC surface. (*P<0.05 Mann–Whitney test n=4) error bars represent standard error mean (s.e.m.). (c) HEK293T transfected with CLEC14A domain deletions were lysed and far western blotted with MMRN2FL under non-reduced conditions. Upon detection of the MMRN2FL His tag, MMRN2 binding was observed in all mutants except those lacking the CTLD or sushi domains. Probing with anti-CLEC14A antibodies was included to show expression of each mutant protein. (d) Flow cytometry analysis of HEK293T transfected with CLEC14A wild-type (wt) with a GFP tag, chimera 1 CLEC14ATHBD(CTLD) or chimera 2 CLEC14ATHBD(sushi). All of the CLEC14A monoclonal antibodies and MMRN2495–674 bound to CLEC14A wt. None of the CLEC14A monoclonal antibodies nor the MMRN2495–674 fragment bound to chimera 1 CLEC14ATHBD(CTLD) except modest binding with C2. All antibodies except C2 bound to chimera 2 CLEC14ATHBD(sushi). The MMRN2495–674 fragment could also bind chimera 2 CLEC14ATHBD(sushi). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/28671670), licensed under a CC-BY license. Not internally tested by R&D Systems.

MMRN2 binds to CLEC14A in the C-type lectin domain. (a) CLEC14A-ECD-Fc pull-downs were pre-incubated with either PBS, mouse control IgG, or CLEC14A monoclonal antibodies C1–5 and used to pull down MMRN2FL from HEK293T-transfected lysates. C1, C4 and C5 blocked MMRN2 enrichment, whereas C2 and C3 did not. (b) Flow cytometry analysis of CLEC14A-ECD-Fc binding to HUVEC surface. CLEC14A-ECD-Fc was pre-incubated with the same blocking conditions as in pull-downs. C1, C4 and C5 significantly blocked CLEC14A-ECD-Fc binding to HUVEC surface. (*P<0.05 Mann–Whitney test n=4) error bars represent standard error mean (s.e.m.). (c) HEK293T transfected with CLEC14A domain deletions were lysed and far western blotted with MMRN2FL under non-reduced conditions. Upon detection of the MMRN2FL His tag, MMRN2 binding was observed in all mutants except those lacking the CTLD or sushi domains. Probing with anti-CLEC14A antibodies was included to show expression of each mutant protein. (d) Flow cytometry analysis of HEK293T transfected with CLEC14A wild-type (wt) with a GFP tag, chimera 1 CLEC14ATHBD(CTLD) or chimera 2 CLEC14ATHBD(sushi). All of the CLEC14A monoclonal antibodies and MMRN2495–674 bound to CLEC14A wt. None of the CLEC14A monoclonal antibodies nor the MMRN2495–674 fragment bound to chimera 1 CLEC14ATHBD(CTLD) except modest binding with C2. All antibodies except C2 bound to chimera 2 CLEC14ATHBD(sushi). The MMRN2495–674 fragment could also bind chimera 2 CLEC14ATHBD(sushi). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/28671670), licensed under a CC-BY license. Not internally tested by R&D Systems.

MMRN2 binds to CLEC14A in the C-type lectin domain. (a) CLEC14A-ECD-Fc pull-downs were pre-incubated with either PBS, mouse control IgG, or CLEC14A monoclonal antibodies C1–5 and used to pull down MMRN2FL from HEK293T-transfected lysates. C1, C4 and C5 blocked MMRN2 enrichment, whereas C2 and C3 did not. (b) Flow cytometry analysis of CLEC14A-ECD-Fc binding to HUVEC surface. CLEC14A-ECD-Fc was pre-incubated with the same blocking conditions as in pull-downs. C1, C4 and C5 significantly blocked CLEC14A-ECD-Fc binding to HUVEC surface. (*P<0.05 Mann–Whitney test n=4) error bars represent standard error mean (s.e.m.). (c) HEK293T transfected with CLEC14A domain deletions were lysed and far western blotted with MMRN2FL under non-reduced conditions. Upon detection of the MMRN2FL His tag, MMRN2 binding was observed in all mutants except those lacking the CTLD or sushi domains. Probing with anti-CLEC14A antibodies was included to show expression of each mutant protein. (d) Flow cytometry analysis of HEK293T transfected with CLEC14A wild-type (wt) with a GFP tag, chimera 1 CLEC14ATHBD(CTLD) or chimera 2 CLEC14ATHBD(sushi). All of the CLEC14A monoclonal antibodies and MMRN2495–674 bound to CLEC14A wt. None of the CLEC14A monoclonal antibodies nor the MMRN2495–674 fragment bound to chimera 1 CLEC14ATHBD(CTLD) except modest binding with C2. All antibodies except C2 bound to chimera 2 CLEC14ATHBD(sushi). The MMRN2495–674 fragment could also bind chimera 2 CLEC14ATHBD(sushi). Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/28671670), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, Flow, IHC, CyTOF-ready, ICC/IF
• Clonality: Polyclonal
• Host: Sheep
• Conjugate: Unconjugated
• Concentration: LYOPH

CLEC14A Antibody [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human CLEC14A
  Glu22-Ala397
  Accession # Q86T13
• Specificity: Detects human CLEC14A in direct ELISAs and Western blots. In direct ELISAs and Western blots, less than 1% cross‑reactivity with recombinant human (rh) CLEC1, rhCLEC2, rhCLEC3B, and rhCLEC10A is observed.
• Source: N/A
• Isotype: IgG
• Clonality: Polyclonal
• Host: Sheep
• Gene: CLEC14A
• Purity Statement: Antigen Affinity-purified

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Flow Cytometry 2.5 ug/10^6 cells
  • Immunocytochemistry 5-15 ug/mL
  • 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 CLEC14A Antibody [Unconjugated]

• C14orf27
• CEG1
• chromosome 14 open reading frame 27
• CLEC14A
• ClECT and EGF-like domain containing protein
• C-type lectin domain family 14 member A
• C-type lectin domain family 14, member A
• EGF R5
• EGFR5
• EGFR-5
• Epidermal growth factor receptor 5

Background

CLEC14A (C-type lectin domain family 14 member A; also EGFR5) is a 51 kDa (predicted) member of the C-type lectin domain family of proteins. It is a type I transmembrane protein, apparently expressed in brain and about which little is known. Mature human CLEC14A is 469 amino acids in length. It contains a 376 aa extracellular region (aa 22-397) and a 72 aa cytoplasmic domain. The extracellular region shows one C-type lectin like domain (aa 32-175) and an EGF-like region (aa 245-287). Over aa 22-397, human CLEC14A shares 66% and 81% aa identity with mouse and canine CLEC14A, respectively.

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 CLEC14A Antibody (AF4968)(8)

Publications using AF4968

• X Zhuang, F Maione, J Robinson, M Bentley, B Kaul, K Whitworth, N Jumbu, E Jinks, J Bystrom, P Gabriele, E Garibaldi, E Delmastro, Z Nagy, D Gilham, E Giraudo, R Bicknell, SP Lee CAR T cells targeting tumor endothelial marker CLEC14A inhibit tumor growth JCI Insight, 2020-10-02;5(19):. 2020-10-02 [PMID: 33004686] (Flow Cytometry, IHC, Human, Mouse)
• J Robinson, K Whitworth, E Jinks, Z Nagy, R Bicknell, SP Lee An evaluation of the tumour endothelial marker CLEC14A as a therapeutic target in solid tumours J Pathol Clin Res, 2020-07-21;0(0):. 2020-07-21 [PMID: 32696621] (IHC, Human)
• TK Kim, CS Park, J Jang, MR Kim, HJ Na, K Lee, HJ Kim, K Heo, BC Yoo, YM Kim, JW Lee, SJ Kim, ES Kim, DY Kim, K Cha, TG Lee, S Lee Inhibition of VEGF-dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a Mol Oncol, 2018-01-26;0(0):. 2018-01-26 [PMID: 29316206] (Flow Cytometry, Human)
• J Jang, MR Kim, TK Kim, WR Lee, JH Kim, K Heo, S Lee CLEC14a-HSP70-1A interaction regulates HSP70-1A-induced angiogenesis Sci Rep, 2017-09-06;7(1):10666. 2017-09-06 [PMID: 28878328] (Flow Cytometry, Western Blot, Human)
• Noy P, Swain R, Khan K, Lodhia P, Bicknell R Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2). FASEB J, 2016-03-03;30(6):2311-23. 2016-03-03 [PMID: 26939791] (Western Blot, Human)
• Noy P, Lodhia P, Khan K, Zhuang X, Ward D, Verissimo A, Bacon A, Bicknell R Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth. Oncogene, 2015-03-09;34(47):5821-31. 2015-03-09 [PMID: 25745997] (Western Blot, Human)
• Ki , M K, Jeoung , M H, Choi , J R, Rho , S-S, Kwon , Y-G, Shim , H, Chung , J, Hong , H J, Song , B D, Lee , S Human antibodies targeting the C-type lectin-like domain of the tumor endothelial cell marker clec14a regulate angiogenic properties in vitro. Oncogene, 2013-05-06;32(48):5449-57. 2013-05-06 [PMID: 23644659] (Flow Cytometry, Human)
• Mura M, Swain RK, Zhuang X Identification and angiogenic role of the novel tumor endothelial marker CLEC14A. Oncogene, 2011-06-27;31(3):293-305. 2011-06-27 [PMID: 21706054] (ICC, IHC-P, Neutralization, Human)

Bioinformatics

• Gene Symbol: CLEC14A
• Uniprot:
  • Q86T13()