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Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody

Catalog #: AF2480
14 Citations Datasheet / COA / SDS
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AF2480
AF2480-SP
Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) by Western Blot.
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Citations (14)
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Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody Summary

Species Reactivity
Human, Mouse
Specificity
Detects human and mouse HGF R/c-MET when phosphorylated at Y1234/Y1235 in Western blots.
Source
Polyclonal Rabbit IgG
Purification
Antigen Affinity-purified
Immunogen
Phosphopeptide containing human HGF R/c-MET Y1234/1235 sites
Formulation
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.
Label
Unconjugated

Applications

Recommended Concentration
Sample
Western Blot
0.5 µg/mL
See below
Simple Western
5 µg/mL
See below
Immunohistochemistry
5-15 µg/mL
See below
CyTOF-reported
Brodie, T.M. et al. (2018) Cytometry PartA. 93: 406. Ready to be labeled using establishedconjugation methods. No BSA or other carrier proteins that could interfere withconjugation.
 
Immunocytochemistry
5-15 µg/mL
See below
Intracellular Staining by Flow Cytometry
2.5 µg/106 cells
See below

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Scientific Data

Western Blot Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) antibody by Western Blot. View Larger

Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) by Western Blot. Western blot shows Goat Anti-Human HGF R/c-MET Antigen Affinity-purified Polyclonal Antibody (Catalog # AF276) immunoprecipitate of MDA-MB-468 human breast cancer cell line untreated (-) or treated (+) with 100 µM pervanadate (PV) for 10 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-HGF R/c-MET (Y1234/Y1235) at approximately 145 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Immunocytochemistry HGF R/c-MET antibody in SH-4 Human Cell Line by Immunocytochemistry (ICC). View Larger

HGF R/c‑MET in SH-4 Human Cell Line. HGF R/c-MET was detected in immersion fixed SH-4 human melanoma cell line stimulated with HGF (left panel; positive staining) and non-stimulated (right panel; negative staining) using Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Immunohistochemistry HGF R/c-MET antibody in Mouse Embryo by Immunohistochemistry (IHC-Fr). View Larger

HGF R/c-MET in Mouse Embryo. HGF R/c-MET was detected in immersion fixed frozen sections of mouse embryo (13 d.p.c.) using Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Immunohistochemistry HGF R/c-MET antibody in Human Renal Cell Carcinoma Tissue by Immunohistochemistry (IHC-P). View Larger

HGF R/c‑MET in Human Renal Cell Carcinoma Tissue. HGF R/c-MET was detected in immersion fixed paraffin-embedded sections of human renal cell carcinoma tissue using Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Intracellular Staining by Flow Cytometry Detection of HGF R/c-MET antibody in pervanadate-treated MCF-7 Human Cell Line antibody by Flow Cytometry. View Larger

Detection of HGF R/c‑MET in pervanadate-treated MCF‑7 Human Cell Line by Flow Cytometry. MCF-7 human breast cancer cell line was unstimulated (light orange open histogram) or treated with 100 µM pervanadate for 10 minutes (dark orange filled histogram), then stained with Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480), or control antibody (Catalog # AB-105-C, blue open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.

Simple Western Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) antibody by Simple Western<SUP>TM</SUP>. View Larger

Detection of Human Phospho-HGF R/c‑MET (Y1234/Y1235) by Simple WesternTM. Simple Western lane view shows lysates of MDA‑MB‑468 human breast cancer cell line untreated (-) or treated (+) with 100 µM Pervanadate (PV) for 10 minutes, loaded at 0.2 mg/mL. A specific band was detected for HGF R/c‑MET at approximately 156 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Flow Cytometry Detection of Human HGFR/c-MET by Flow Cytometry View Larger

Detection of Human HGFR/c-MET by Flow Cytometry Amuvatinib suppresses MET receptor tyrosine kinase activity. (A) Flow cytometry analysis of p-MET (Tyr1234/1235) levels in U266 cells were treated for 24 hrs with DMSO (gray shaded) and 25 μM amuvatinib (dark black line). (B) Quantification of phospho (black bars) and total (gray bars) MET staining in U266 cells treated with the indicated concentrations of amuvatinib (Rx) from quadruplet experiments as in (A). (C) U266 cells were serum starved and treated with the indicated concentrations of amuvatinib or DMSO and stimulated with 50 ng/ml HGF for 15 min. Cell lysates were subjected to immunoblot analysis to assess MET (Y1349) phosphorylation. (D) The 140 kDa (solid bars) and the 170 kDa (speckled bars) phospho (black bars) and total (gray bars) MET bands from triplicate experiment as in (C) were quantitated and normalized to GAPDH levels. The results are presented as percentages of the HGF-stimulated DMSO controls. Data are representative of three independent experiments and presented as Mean ± SEM, n = 3, *P < 0.05, **P < 0.01. Image collected and cropped by CiteAb from the following publication (https://jhoonline.biomedcentral.com/articles/10.1186/1756-8722-6-92), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reconstitution Calculator

Reconstitution Calculator

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Preparation and Storage

Reconstitution
Reconstitute at 0.2 mg/mL in sterile PBS.
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Shipping
Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.
Stability & 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.

Background: HGFR/c-MET

HGF R, also known as Met (from N-methyl-N’-nitro-N-nitrosoguanidine induced), is a glycosylated receptor tyrosine kinase that plays a central role in epithelial morphogenesis and cancer development. HGF R is synthesized as a single chain precursor which undergoes cotranslational proteolytic cleavage. This generates a mature HGF R that is a disulfide-linked dimer composed of a 50 kDa extracellular alpha chain and a 145 kDa transmembrane beta chain (1, 2). The extracellular domain (ECD) contains a seven bladed beta -propeller sema domain, a cysteine-rich PSI/MRS, and four Ig-like E-set domains, while the cytoplasmic region includes the tyrosine kinase domain (3, 4). Proteolysis and alternate splicing generate additional forms of human HGF R which either lack of the kinase domain, consist of secreted extracellular domains, or are deficient in proteolytic separation of the alpha and beta chains (5‑7). The sema domain, which is formed by both the alpha and beta chains of HGF R, mediates both ligand binding and receptor dimerization (3, 8). Ligand-induced tyrosine phosphorylation in the cytoplasmic region activates the kinase domain and provides docking sites for multiple SH2-containing molecules (9, 10). HGF stimulation induces HGF R downregulation via internalization and proteasome-dependent degradation (11). In the absence of ligand, HGF R forms noncovalent complexes with a variety of membrane proteins including CD44v6, CD151, EGF R, Fas, Integrin alpha 6/ beta 4, Plexins B1, 2, 3, and MSP R/Ron (12‑19). Ligation of one complex component triggers activation of the other, followed by cooperative signaling effects (12‑19). Formation of some of these heteromeric complexes is a requirement for epithelial cell morphogenesis and tumor cell invasion (12, 16, 17). Paracrine induction of epithelial cell scattering and branching tubulogenesis results from the stimulation of HGF R on undifferentiated epithelium by HGF released from neighboring mesenchymal cells (20). Genetic polymorphisms, chromosomal translocation, overexpression, and additional splicing and proteolytic cleavage of HGF R have been described in a wide range of cancers (1). Within the ECD, human HGF R shares 86%‑88% aa sequence identity with canine, mouse, and rat HGF R.

References
  1. Birchmeier, C. et al. (2003) Nat. Rev. Mol. Cell Biol. 4:915.
  2. Corso, S. et al. (2005) Trends Mol. Med. 11:284.
  3. Gherardi, E. et al. (2003) Proc. Natl. Acad. Sci. 100:12039.
  4. Park, M. et al. (1987) Proc. Natl. Acad. Sci. 84:6379.
  5. Crepaldi, T. et al. (1994) J. Biol. Chem. 269:1750.
  6. Prat, M. et al. (1991) Mol. Cell. Biol. 12:5954.
  7. Rodrigues, G.A. et al. (1991) Mol. Cell. Biol. 11:2962.
  8. Kong-Beltran, M. et al. (2004) Cancer Cell 6:75.
  9. Naldini, L. et al. (1991) Mol. Cell. Biol. 11:1793.
  10. Ponzetto, C. et al. (1994) Cell 77:261.
  11. Jeffers, M. et al. (1997) Mol. Cell. Biol. 17:799.
  12. Orian-Rousseau, V. et al. (2002) Genes Dev. 16:3074.
  13. Klosek, S.K. et al. (2005) Biochem. Biophys. Res. Commun. 336:408.
  14. Jo, M. et al. (2000) J. Biol. Chem. 275:8806.
  15. Wang, X. et al. (2002) Mol. Cell 9:411.
  16. Trusolino, L. et al. (2001) Cell 107:643.
  17. Giordano, S. et al. (2002) Nat. Cell Biol. 4:720.
  18. Conrotto, P. et al. (2004) Oncogene 23:5131.
  19. Follenzi, A. et al. (2000) Oncogene 19:3041.
  20. Sonnenberg, E. et al. (1993) J. Cell Biol. 123:223.
Long Name
Hepatocyte Growth Factor Receptor
Entrez Gene IDs
4233 (Human); 17295 (Mouse)
Alternate Names
AUTS9; cMET; c-MET; EC 2.7.10; EC 2.7.10.1; hepatocyte growth factor receptor; HGF R; HGF receptor; HGF/SF receptor; HGFR; Met (c-Met); met proto-oncogene (hepatocyte growth factor receptor); met proto-oncogene tyrosine kinase; MET; oncogene MET; Proto-oncogene c-Met; RCCP2; Scatter factor receptor; SF receptor; Tyrosine-protein kinase Met

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Citations for Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

14 Citations: Showing 1 - 10
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  1. MET Receptor Tyrosine Kinase Inhibition Reduces Interferon-Gamma (IFN-gamma )-Stimulated PD-L1 Expression through the STAT3 Pathway in Melanoma Cells
    Authors: Song, KY;Han, YH;Roehrich, H;Brown, ME;Torres-Cabala, C;Giubellino, A;
    Cancers
    Species: Human
    Sample Types: Cell Lysates
    Applications: Western Blot
  2. Genetic Ablation of the MET Oncogene Defines a Crucial Role of the HGF/MET Axis in Cell-Autonomous Functions Driving Tumor Dissemination
    Authors: Modica, C;Cortese, M;Bersani, F;Lombardi, AM;Napoli, F;Righi, L;Taulli, R;Basilico, C;Vigna, E;
    Cancers
    Species: Xenograft
    Sample Types: Whole Tissue
    Applications: Immunohistochemistry
  3. A receptor-antibody hybrid hampering MET-driven metastatic spread
    Authors: Chiara Modica, Cristina Basilico, Cristina Chiriaco, Nicla Borrelli, Paolo M. Comoglio, Elisa Vigna
    Journal of Experimental & Clinical Cancer Research
  4. Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation
    Authors: AR Virzì, A Gentile, S Benvenuti, PM Comoglio
    Proc. Natl. Acad. Sci. U.S.A., 2018-09-17;0(0):.
    Species: Mouse
    Sample Types: Whole Tissue
    Applications: IHC
  5. Pulmonary pericytes regulate lung morphogenesis
    Authors: K Kato, R Diéguez-Hu, DY Park, SP Hong, S Kato-Azuma, S Adams, M Stehling, B Trappmann, JL Wrana, GY Koh, RH Adams
    Nat Commun, 2018-06-22;9(1):2448.
    Species: Mouse
    Sample Types: Cell Lysates
    Applications: Western Blot
  6. Antiproliferative effects of the CDK6 inhibitor PD0332991 and its effect on signaling networks in gastric cancer cells
    Authors: D Wang, Y Sun, W Li, F Ye, Y Zhang, Y Guo, DY Zhang, J Suo
    Int. J. Mol. Med., 2018-02-06;0(0):.
    Species: Human
    Sample Types: Cell Lysates
    Applications: Protein Array
  7. Novel potential predictive markers of sunitinib outcomes in long-term responders versus primary refractory patients with metastatic clear-cell renal cell carcinoma
    Authors: Javier Puente, Nuria Laínez, Marta Dueñas, María José Méndez-Vidal, Emilio Esteban, Daniel Castellano et al.
    Oncotarget
  8. Dual MET/EGFR therapy leads to complete response and resistance prevention in a MET-amplified gastroesophageal xenopatient cohort
    Oncogene, 2016-08-15;0(0):.
    Species: Human
    Sample Types: Whole Tissue
    Applications: IHC-P
  9. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer
    Authors: Gaofeng Fan, Siwei Zhang, Yan Gao, Peter A. Greer, Nicholas K. Tonks
    Genes & Development
  10. Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines.
    Authors: Phillip C, Zaman S, Shentu S, Balakrishnan K, Zhang J, Baladandayuthapani V, Taverna P, Redkar S, Wang M, Stellrecht C, Gandhi V
    J Hematol Oncol, 2013-12-10;6(0):92.
    Species: Human
    Sample Types: Whole Cells
    Applications: Flow Cytometry
  11. ETS2 Mediated Tumor Suppressive Function and MET Oncogene Inhibition in Human Non–Small Cell Lung Cancer
    Authors: Mohamed Kabbout, Melinda M. Garcia, Junya Fujimoto, Diane D. Liu, Denise Woods, Chi-Wan Chow et al.
    Clinical Cancer Research
  12. Protein signatures for classification and prognosis of gastric cancer a signaling pathway-based approach.
    Authors: Wang D, Ye F, Sun Y, Li W, Liu H, Jiang J, Zhang Y, Liu C, Tong W, Gao L, Sun Y, Zhang W, Seetoe T, Lee P, Suo J, Zhang DY
    Am. J. Pathol., 2011-08-18;179(4):1657-66.
    Species: Human
    Sample Types: Tissue Homogenates
    Applications: Array Development
  13. Alternative proteolytic processing of hepatocyte growth factor during wound repair.
    Authors: Buchstein N, Hoffmann D, Smola H, Lang S, Paulsson M, Niemann C, Krieg T, Eming SA
    Am. J. Pathol., 2009-04-23;174(6):2116-28.
    Species: Human
    Sample Types: Whole Tissue
    Applications: IHC-Fr
  14. Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells.
    Authors: Kawaguchi K, Murakami H, Taniguchi T, Fujii M, Kawata S, Fukui T, Kondo Y, Osada H, Usami N, Yokoi K, Ueda Y, Yatabe Y, Ito M, Horio Y, Hida T, Sekido Y
    Carcinogenesis, 2009-04-20;30(7):1097-105.
    Species: Human
    Sample Types: Whole Tissue
    Applications: IHC-P

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