Tie1-751 interacts with Tie1 and Tie2. (a) Specific binding of 125I-Tie1-751(6His) to human umbilical vein endothelial cells (HUVEC). Nonspecific binding was determined in the presence of 100-fold excess of unlabelled Tie1-751 and was subtracted from the total binding. CPM, counts per minute; Kd, dissociation constant. (b) Binding of 125I-Tie1-751(6His) to HUVEC was competed by increasing amounts of cold Tie1-751. Data are the mean ± standard error of the mean. (c) Binding of Tie1-751(6His) to HUVEC. At the end of binding, cells were treated with or without the cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), immunoprecipitated using a C-terminal-specific anti-Tie1 (top panel) or anti-Tie2 (middle panel) antibody, and were analyzed by western blotting using anti-His antibody. To confirm equal loading, cell lysates were blotted with anti-Tie1 antibody (bottom panel). IP, Immunprecipitation; WB, Western blot.