Protean 3 mini gel apparatus

After the protein lysates were prepared from homogenizing the frozen tumor tissues, the bicinchoninic acid protein assay (Santa Cruz, CA, USA) was used to detect the protein concentration. Thirty micrograms of protein in the loading buffer were incubated at 95°C for 5 min, cooled, and then loaded per lane. Gel electrophoresis was performed on a Protean III mini-gel apparatus (Bio-Rad, Hercules, CA, USA) using 8% gel with 0.1% (w/v) SDS under a constant current of 22 mA and then transferred to nitrocellulose membranes (Dingguo Biotechnology Company, Beijing, China) for 2.5 h. The membranes were then blocked for 2 h at room temperature with 5% milk in Tris-Buffered Saline Tween (TBST: 10 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% Tween-20). Membranes were incubated with primary antibody dilution (VEGF antibody from Abclonal No. A0280, 1:800; β-actin antibody from Santa, 1:1000) overnight at 4°C. After washing, the membranes were incubated with their corresponding secondary antibody (1:3000) at room temperature for 2.5 h. After the proteins were detected with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA), the densitometric intensity was measured with a GS-800 densitometer (Bio-Rad) and normalized against internal control β-actin.