Figure 1.

ExTek.6His blocks Ang binding and Ang-stimulated cellular responses. ExTek.6His blocks binding of Ang1* (A) and Ang2 (B) to immobilized Tie2-Fc. Binding of Ang1* and Ang2 (800 ng/ml) to immobilized Tie2-Fc was done in the presence of increasing ExTek.6His concentrations ranging from 0 to 50 μg/ml. The closely related Tie1-Fc (50 μg/ml) was used to verify specificity of inhibition of ligand binding to the immobilized Tie2-Fc. Data are expressed as the means ± SD of three replicate experiments. ExTek.6His blocks Tie2 phosphorylation induced by Ang1* in endothelial cells (C). EAhy926 cells were serum-starved overnight followed by stimulation with Ang1* (150 ng/ml) plus the indicated amount of ExTek.6His or control protein, ExFms.6His, for 8 min. Tie2 and Tie2 phosphorylation levels then were detected by immunoprecipitation/Western blot (anti-Tie2 and anti-P-tyrosine, respectively). ExTek.6His blocks Ang1*-mediated cell survival/proliferation (D). Tie2/TrkC expressing cells were incubated in serum-free medium that contained increasing concentrations of ExTek.6His and a constant amount of Ang1* (400 ng/ml). The vital dye MTT was added to the cultures after 2 days of treatment, and the amount of MTT-derived blue product in the cell lysates was determined spectrophotometrically and used as an index of live cells present. All values were corrected by subtracting the value of a culture with no added factors and represent the means ± SD of three replicate wells.