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.
KDR
VEGFR2, FLK1, VEGF-R2
HUE
Endogenous
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
Freiburg, Germany
More information can be found on our website Cellular Phosphorylation Assay Services.
Primary reference compound IC50 for VEGF-R2
Whereas Sunitinib blocks VEGF-R2 as a broad kinase inhibitor, PTK-787[1] 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.
[1] Wood, JM et al. (2000) Cancer Res. 60(8):2178-89.
Additional validation data
Whereas Sunitinib blocks VEGF-R2 as a broad kinase inhibitor, PTK-787[1] 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.
[1] Wood, JM et al. (2000) Cancer Res. 60(8):2178-89.