Phospho vegf receptor 2 tyr1175

Western blot analysis was performed with 8% SDS/PAGE using standard methods. In brief, fresh nitrogen-frozen tumor tissues were placed directly into RIPA lysis buffer (Boston BioProducts Inc, MA Cat# BP-115) contained Complete ULTRA tablets, Mini EDTA-free (Roche Diagnostics, IN Cat# 05892791001), and PhosStop (Roche Diagnostics, IN Cat# 04906837001), incubated at 4 °C for 15 min, and then centrifuged at 14,000 rpm (~ 18,000×g), 4 °C, for 10 min for protein extraction. Approximately 20 μg of denatured protein per sample lysates were loaded on SDS/PAGE, separated by electrophoresis and transferred onto a polyvinylidene difluoride membrane (PVDF) using wet electroblotting transfer at 200 mA for 2.5 h. Non-specific binding was blocked with 5% non-fat milk for 1 h. Membranes were incubated overnight at 4 °C with the specific primary antibodies, followed by peroxidase-conjugated anti-rabbit HRP secondary antibodies. The following antibodies were used: Phospho-VEGF Receptor 2 Tyr1175 (1:200, Cell Signaling Technology, MA Cat# 2478), VEGF Receptor 2 (1:1000, Cell Signaling Technology, MA Cat# 2479), and β-Actin (1:5000, Sigma, MO Cat# A5441). Image J software was used for densitometric analysis. The ratio area pixels of the phosphor-VEGFR2 to total VEGFR2 was calculated.