Fig. 2.

Suppression of Tie1 expression differentially affects binding of Ang1 and Ang2 to Tie2 in endothelial cells. (A) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of control vehicle (C) or 200 ng/ml Ang2 (A2) for 30 min as indicated followed by cross-linking with the cell-impermeable cross-linker DTSSP. 20 mM Tris was added to quench cross-linking before washing and cell lysis. Ang2 was immunoprecipitated and immunoprecipitates or whole cell lysates (Wcl) were resolved by SDS/PAGE. Tie2 bound to Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were transfected with siRNA directed against Tie1 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang1 (A1) or Ang2 (A2) for 30 min as indicated. Cross-linking, quenching and immunoprecipitation were performed as above and Tie2 bound to Ang1 and Ang2 and Tie1 and Tie2 in whole cell lysates were detected by immunoblotting as indicated. The effect of suppression of Tie1 expression by siRNA on Ang1 and Ang2 binding to Tie2 on cells was determined in three independent experiments by immunoblotting and densitometric quantification of blots. Data are presented as means and SEM. ⁎ indicates Ang1 binding to Tie2 was significantly increased by loss of Tie1 (p < 0.05, Student's t test).