Fig. 1. Ang1 and Ang2 activate Tie2 in a concentration-dependent manner. (A,B) HUVECs were stimulated with the indicated concentrations of Ang1 pre-clustered with 10 µg/ml anti-polyhistidine antibody (A) or Ang2 (B) for 15 minutes at 37°C. Top panels are representative immunoblots. Intensities of Tie2 tyrosine phosphorylation were quantified by densitometry and plotted versus Ang1 or Ang2 concentration (lower panels). The value at 800 ng/ml was designated 100%. (C) Comparison of Tie2 tyrosine phosphorylation when HUVECs are stimulated with Ang1 (800 ng/ml), Ang1 (800 ng/ml) pre-clustered with the anti-polyhistidine (anti-his) antibody (10 µg/ml), Ang2 (800 ng/ml), or the anti-polyhistidine antibody (10 µg/ml) alone. (D) EA.hy926 cells were stimulated with Ang1 (800 ng/ml, pre-clustered) or Ang2 (800 ng/ml) for 15 minutes at 37°C. Tie2 was immunoprecipitated and immunoblotted with an anti-phospho Tyr1102 and Tyr1108 antibody (top panel) or an anti-Tie2 antibody (lower panel).