VEGF-R3 Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The receptor tyrosine kinase VEGF-R3 is mainly expressed in lymphatic endothelial cells. Binding of vascular endothelial growth factor C or D activates receptor autophosphorylation via the mechanism of receptor dimerization. In many tumors overexpression of VEGF-R3 or its ligand VEGF C appears to play an important role in tumor angiogenesis and lymphatic metastasis.
FLT4
VEGFR3, PCL, VEGF-R3
MEF
Transfected
The cellular VEGF-R3 phosphorylation assay was generated on a mouse embryonal fibroblast (MEF) background. Cells were transfected to express a full-length VEGF-R3 protein. After clonal selection a cell line expressing high levels of VEGF-R3 was obtained. Overexpression of the kinase results in a constitutive, ligand-independent autophosphorylation of the receptor which is potently inhibited in the presence of cognate VEGF-R3 inhibitors such as PTK787 or Sunitinib (see Fig. 1). Phospho-VEGF-R3 levels are quantified by Sandwich-ELISA technique.
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
Freiburg, Germany
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Primary reference compound IC50 for VEGF-R3
PTK787 and Sunitinib are potent inhibitors of the phospho-VEGF-R3 signal found in the described cells. With both compounds highly reproducible IC50 values were generated in the cellular VEGF-R3 assay. The graph show representative results.
Additional validation data IC50 for VEGF-R3
PTK787 and Sunitinib are potent inhibitors of the phospho-VEGF-R3 signal found in the described cells. With both compounds highly reproducible IC50 values were generated in the cellular VEGF-R3 assay. The graph show representative results.