HUVECs were purchased from Lonza. The cells were confirmed to be mycoplasma-negative (MycoAlert Detection Kit, Lonza), cultured in endothelial growth medium (EGM2-MV, Lonza), and incubated in a humidified atmosphere of 5% CO2 at 37°C. For TIE2 immunoprecipitation, HUVECs were seeded into 100-mm culture dishes and grown to confluence. HUVECs were starved for 6 hours and then incubated with human ANGPT2 (1 μg/ml; R&D Systems) in the presence of ABTAA for 30 min. Cells were rinsed once with cold PBS and lysed in cold complete lysis-M buffer (Roche) containing protease and phosphatase inhibitors (Roche). The lysates were centrifuged at 14,000 g at 4°C for 15 min, and supernatants were subjected to immunoprecipitation with anti-TIE2 antibody (R&D systems). The immunoprecipitates were incubated with 20 μl of Dynabeads Protein G (Life Technologies) for 2 hours. Beads with immunoprecipitates were washed three times with cold lysis buffer, heated in Laemmli sample buffer at 95°C for 5 min, subjected to SDS-PAGE on 4 to 15% Mini-PROTEAN TGX Precast Gels (Bio-Rad), transferred to nitrocellulose membrane (Invitrogen), and probed with horseradish peroxidase (HRP)–conjugated anti–phosphotyrosine 4G10 antibody (Millipore). The blots were developed using the ECL Western blotting detection kit (GE Healthcare) and visualized with Amersham Imager 600 (GE Healthcare). The membranes were stripped and reprobed with an anti-TIE2 (Millipore) antibody. The primary antibodies for pAKT (S473), AKT, pERK (T202/Y204), and ERK (all from Cell Signaling) were used. Secondary HRP-conjugated antibodies (Bio-Rad) were used for signal detection.

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