VEGF-R2 Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
VEGF-R2 is almost exclusively expressed on endothelial cells. It transduces the action of the well characterized pro-angiogenic vascular endothelial growth factor (VEGF-A). In pathological situations that involve neovascularization as well as enhanced vascular permeability the VEGF/VEGF-R2 signaling is responsible for activated endothelium at the tumor site. Today, VEGF-R2 is a validated target for cancer therapeutics addressing solid tumors with induced blood vessel formation.
VEGFR2, FLK1, VEGF-R2
Immortalized human umbilical vein endothelial cells (HUE) are known to overexpress human VEGF-R2. Stimulation of these cells with its physiological ligand VEGF-A, results in a robust receptor autophosphorylation. Compounds are preincubated before cell stimulation to allow thorough target binding. Stimulation conditions are optimized to determine dose-related inhibition of the phospho-VEGF-R2 signal, which is subsequently quantified by Sandwich ELISA technique. The assay is validated based on known inhibitors of VEGF-R2 kinase activity (see Fig. 1).
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
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Reference compound IC50 for VEGF-R2
Whereas Sunitinib blocks VEGF-R2 as a broad kinase inhibitor, PTK-787 is known to inhibit the VEGF-induced phosphoVEGF-R2 signal in a highly specific manner. Both compounds were included in the validation process and the cellular VEGFR2 assay generated highly reproducible IC50 values. The graphs show representative results.
 Wood, JM et al. (2000) Cancer Res. 60(8):2178-89.