Anti vegfr

Protein was extracted from the cells by using a lysis buffer containing protease inhibitor (KeyGen BioTech) and was quantified with a bicinchoninic acid kit (KeyGen BioTech). Thirty micrograms of protein extracts were separated using 10% sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) gel before transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk for 1 h, the membranes were then incubated with primary antibodies at 4°C overnight: anti-VEGFA (1:1,000; Proteintech, Chicago, IL, USA), GAPDH (1:5,000; Proteintech), anti-VEGFR (1:1,000; Cell Signaling Technology, Beverly, MA, USA), anti-p-VEGFR2 (1:1,000; Cell Signaling Technology), and anti-p-Akt (1:1,000; Cell Signaling Technology). Next day, the membranes were incubated with secondary antibody for 1 h at room temperature. The protein bands were detected using an enhanced chemiluminescence reagent kit (KeyGen BioTech).

Western blot analysis was carried out according to standard procedures using enhanced-chemiluminescence detection (GE Life Science, Chicago, IL, USA). Tumor tissue lysates were prepared using an electric homogenizer for 30 seconds after the addition of lysis buffer. The following antibodies were used: anti-β-actin, anti-acetyl-CoA carboxylase (ACC), anti-phospho-ACC, anti-phospho-AKT, anti-phospho-BRCA1, anti-caspase-3, anti-caspase-7, anti-β-catenin, anti-phospho-cyclin D1, anti-phospho-GSKα/β, anti-MAPK, anti-phospho-MAPK, anti-PARP, anti-PDK1, anti-phospho-PDK1, anti-PI3K, anti-phospho-PI3K, anti-phospho-S6, anti-phospho-mTOR, anti-phospho-Rb, anti-VEGFR, anti-phospho-VEGFR (all from Cell Signaling Technology, Danvers, MA, USA); anti-β-actin, anti-AKT, anti-AKT1, anti-BRCA1, anti-cyclin D1, anti-Rb, anti-S6, anti-mTOR, anti-α-tubulin (all from Santa Cruz, Dallas, TX, USA); and anti-PCNA (Atlas Antibodies, Bromma, Sweden). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson Immuno Research, West Grove, PA, USA) were used as secondary antibodies as appropriate.