The analyzed cells were lysed as previously described.3 (link) Subsequently, the collected lysate was centrifuged at 4°C at 18,624xg for 15 min and protein concentration was determined by use of the Bradford method. In total, 50 µg protein was loaded and separated by SDS-PAGE and transferred onto Immobilon-P membranes using the iBlot apparatus (Invitrogen; Thermo Fisher Scientific, Inc.). After blocking in 5% fat-free milk, the membrane was incubated with the appropriate diluted primary antibody overnight at 4°C. The primary antibodies were used with the following dilution: Actin (Transgen Biotech Co., Ltd.; 1:5,000), ZNF750 (Abcam; 1:500) and KDR (Abcam; 1:200). Antibody binding was detected with horseradish-peroxidase-conjugated anti-mouse (Sigma-Aldrich; Merck KGaA) or anti-rabbit (Cell Signaling Technology, Inc.) antibodies for 1 h at room temperature and chemiluminescence was measured using a LAS4000 Device Chemiluminescence System (Beijing Sage Creation Science Co., Ltd.). Each experiment was repeated at least 3 times.