Fig. 3.

Ang1 and Ang2 bind differentially to Tie2 in Tie2:Tie1 complexes. (A) Endothelial cells were transfected with siRNA directed against Tie2 or control randomised siRNA (Sc) and cultured for 24 h before addition of 200 ng/ml Ang2 for 30 min 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. Tie1 and Tie2 recovered from immunoprecipitates and in whole cell lysates were detected by immunoblotting as indicated. (B) Endothelial cells were treated with 200 ng/ml Ang1 or 200 ng/ml Ang2 for 30 min before cross-linking, quenching and immunoprecipitation, as above, and Tie1 and Tie2 recovered from immunoprecipitates were detected by immunoblotting as indicated. Tie1 and Tie2 bound to angiopoietins were determined by immunoblotting in three independent experiments. Data is presented as means and SEM of the ratio of Tie1:Tie2 recovered. ⁎ indicates statistically significant difference between Tie1:Tie2 ratio recovered by Ang1 and Ang2 (p < 0.05, Student's t test).