Total protein was extracted from KGN cells and ovarian tissues. Samples (60 – 80 mg of protein) were subsequently separated with 10 %~12.5 % SDS-PAGE gels for two h using a 300-mA electric current. Non-fat milk was used to treat membranes for 2 h at 4 °C before probing overnight with antibodies against HB-EGF (catalog # 15,071; Cell Signaling Technology, USA), p53 (catalog # wL01919; Wanleigio), p21 (catalog # WLH0362; Wanleigio), p16 (catalog # WLH3673; Wanleigio) and β-actin (catalog # 3700; Cell Signaling Technology). After that, membranes were incubated with infrared (IR) fluorescent dye-conjugated secondary antibodies (1:10,000 – 20,000 dilution; LI-COR Biosciences, Lincoln, NE, USA) for 1 h. An LI-COR Biosciences Infrared Imaging System was used to detect blots with the band intensity quantified using LI-COR Biosciences Odyssey 3.0 software. All values were standardized against β-actin expression.
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