Immobilon p transfer polyvinylidene fluoride membrane

After all treatments, the PC-3 cells were harvested. SDS buffer (Beyotime, Shanghai, China) was used to resuspend the cells to obtain the total protein extracts from PC-3 cells and tissue samples in accordance with the manufacturer’s instructions. We used 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to separate the protein, and then transferred it to an Immobilon-P transfer polyvinylidene fluoride membrane (Millipore, Bedford, MA) for 2 h (120 V). We used 5% bovine serum albumin (BSA) prepared with TBS-T buffer to treat the membrane for 120 min at room temperature. Primary antibody anti-NOS1 at a dilution of 1:2000 (Boster, China) and anti-β-actin at a dilution of 1:5000 (Boster, China) containing 1% BSA was used to incubate the membrane for 12 h at 4°C. A secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG) at a dilution of 1: 10 000 (Boster, China) containing 1% BSA was used to incubate the membrane for 6 h at 4°C. HRP substrate (BeyoECL plus A/B, Beyotime, Shanghai, China) was used to visualize the membrane according to the recommendations of the supplier. Each test was repeated in triplicate.