Binding of Tie1-751 to human umbilical vein endothelial cells
For cell surface binding of 125I-Tie1-751(6His), HUVEC were seeded into a 96-well plate at 1.4 × 104 cell/well in endothelial growth medium-2. Next day, medium was replaced with an ice-cold binding buffer (Hanks' balanced salt solution supplemented with 20 mM Hepes and 0.25% bovine serum albumin, pH 7.5). 125I-Tie1-751 was added to the binding buffer in the presence or absence of unlabeled Tie1-751. Binding was performed at 4°C for 1 hour followed by four washes with ice-cold PBS/0.05% Tween-20. A scintillation cocktail OptiPhase 'SuperMix' (PerkinElmer, Waltham, MA, USA) was added to each well, and the plates were read by Microbeta Trilux (PerkinElmer).
For direct binding of Tie1-751 to transmembrane Tie1 and Tie2, HUVEC were seeded into a six-well plate at 0.5 × 106/well in endothelial growth medium-2. Next day, binding was carried out at 4°C for 1 hour in an ice-cold binding buffer (as above) containing 1 μM purified Tie1-751(6His). At the end of the binding, cells were treated with or without the membrane-impermeable chemical amine-reactive cross-linking agent DTSSP (3,3'-dithiobis [sulfosuccinimidylpropionate] (Pierce Biotechnology Inc., Rockford, IL, USA) at 1 mM for 30 minutes. This treatment was followed by inactivation of 3,3'-dithiobis(sulfosuccinimidylpropionate) with 20 mM Tris buffer, pH 7.5, for 15 minutes. Cells were subsequently lysed and immunoprecipitated using a C-terminal-specific anti-Tie1 or anti-Tie2 antibody. The immunoprecipitated proteins were analyzed by western blotting using anti-His antibody that recognizes the His-tagged Tie1-751.