Ecl exposure system

Cells were harvested and lysed in RIPA buffer with 1% phosphatase and protease inhibitors. Protein content was quantified by BCA protein quantification kits; the same amount (∼30 μg) of proteins was separated and transferred onto NC membranes (Millipore). After blocking membranes for 1 h at room temperature, the NC membranes were incubated with dilutions containing specific primary antibodies overnight at 4℃. Next day, the membrane was washed three times (10 min/each time) with 1 × TBST and incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature. Finally, the membrane was washed three times (10 min/each time) again and visualized using the ECL exposure system (Thermo Fisher Scientific).

LNCap cells were cultured in six-well plates and lysed in RIPA buffer with 1% phosphatase and protease inhibitors. Then, the cell protein contents were quantified via bicinchoninic acid protein quantification kits. The amount (∼50 μg) of proteins was separated and transferred onto NC membranes (Millipore). The blocking membranes used were NC for 1 h. The following primary antibodies were used: rabbit CNDP2 (1:500, PROTEINTECH, catalog number: 14925-1-AP), rabbit SERPINH1 (1:500, HUABIO, R1511-11), and rabbit GAPDH (1:500, Cell Signaling Technology, MA, USA) to incubate overnight at 4°C. On the next day, the membrane was washed three times (10 min/each time) with 1× TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were immersed in ECL exposure system (Thermo Fisher Scientific) to develop luminescence.