Quantity one imaging program

Protein was extracted from the ovarian cancer tissues using radioimmunoprecipitation assay buffer, containing 1% nonyl phenoxypolyethoxylethanol-40, 0.5 sodium deoxycholate and 0.1% sodium dodecyl sulfate. A total of 20 μg protein extract was separated by 10% SDS-PAGE, and electrotransferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% nonfat dry milk and 0.1% Tween^® 20 for 1 h at room temperature with constant agitation, followed by incubation with a primary antibody (1:1,000 dilution) overnight at 4°C. The membranes were washed three times each, for 5 min, with PBST (PBS with Tween), and the membrane was incubated with a secondary horseradish peroxidase-linked antibody (1:2,000 dilution) for 2 h. The membrane was then washed a further three times with PBST, and the bands were visualized by enhanced chemiluminescence, according to the manufacturer’s instructions (Pierce Biotechnology Inc., Rockford, IL, USA). The relative protein expression was normalized to GAPDH (1:1,000 dilution), and expressed as a ratio to the control subjects. The protein bands, including GAPDH, were quantified by densitometry using the Quantity One^® imaging program (Bio-Rad, Hercules, CA, USA).