Detection of Human Phospho-HGF R/|
c-MET (Y1349) by Western Blot. Western blot shows lysates of A431 human epithelial carcinoma 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 Phospho-HGF R/c-MET (Y1349) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3950), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho‑HGF R/c-MET (Y1349) at approximately 145 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
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.