Anti phospho vegf receptor 2

Tumor tissues of the xenograft mice model from different groups were homogenized in RIPA lysis buffer, containing protease inhibitor cocktail EDTA-free, and then were centrifuged at 15,000 rpm for 15 minutes. The supernatants were collected, and the total protein concentration was determined using the bicinchoninic acid (BCA) kit (Chengdu Must Biotechnology Co., Ltd, Chengdu, China). Tumor lysates (20 μg) were subsequently subjected to 12% SDS-PAGE gels and transferred to a nitrocellulose membrane. After blocking with 5% non-fat dry milk at room temperature for 2 hours, the primary antibodies of vascular endothelial growth factor (VEGF, 1:2,000; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-VEGF receptor 2 (Cell Signaling Technology), caspase-3 (1:1,000; Cell Signaling Technology), Bcl-2 (1:1,000; Cell Signaling Technology), Bax (1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Cell Signaling Technology) were added and incubated overnight. Subsequently, the membrane was then washed in TBS+Tween and incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected by enhanced chemiluminescent reagents (Amersham Biosciences, Sydney, NSW, Australia).