Prostate cancer cells were harvested and lysed by RIPA buffer on ice, the supernatant was quantified by BCA protein quantification assay (Bio-Rad, USA). Equal amounts of protein sample were added to 4× sample buffer and boiled for 10 min. The sample was subjected to SDS-PAGE analysis and transferred to nitrocellulose membrane. The membrane was blocked by 5% milk for 2 h at room temperature and incubated with primary antibody at 4°C overnight. The second day, the membrane was washed three times with 1 × TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized by super signal West Pico Stable Peroxide Solution (Thermo Fisher Scientific, USA).

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