Fig. 1.

Binding between Ang2 and Tie2 is not regulated by Tie1 ectodomain cleavage. (A) Endothelial cells were treated with 10 ng/ml PMA or PMA plus 100 μM TAPI-2 before addition of 200 ng/ml Ang2 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 via its His-tag 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. Tie1 ectodomain cleavage is indicated by loss of the higher molecular mass Tie1 immunoreactive band corresponding to surface expressed Tie1 (arrow). (B) Tie2 bound to Ang2 was determined by immunoblotting in three independent experiments. Data is presented as means and SEM. No significant effect of PMA on Tie2 binding was observed (Student's t test).