Protein imager

To extract the protein, the penile spongy tissue was pulverized and added into the radioimmunoprecipitation assay lysis buffer containing enzyme inhibitors (Beyotime Biotechnology). The bicinchoninic acid protein concentration measurement kit (Beyotime Biotechnology) was used to quantify the protein content of supernatants after centrifugation. The supernatant was mixed with a loading buffer and heated to 100 °C to denature the protein. The samples were stored at −20 °C. After electrophoresis, transmembrane, and membrane closure, the polyvinylidene difluoride membranes were incubated with primary antibodies at 4 °C overnight: anti-eNOS antibody (1:1000; Abcam), anti-phospho-eNOS-Ser¹¹⁷⁷ antibody (1:1000; Abcam), and anti-GIT1 antibody (1:100; Santa Cruz Biotechnology). After the membranes were rinsed, the horseradish peroxidase–conjugated secondary antibody (1:1000; Proteintech) was added and incubated at room temperature for 1 hour. The protein strips were presented by the protein imager (Bio-Rad Laboratories) after addition of the enhanced chemiluminescence solution. The grayscale value of the protein strips was quantitated with QuantityOne 4.6 software (Bio-Rad Laboratories).¹⁹ (link)^,22 (link)

Total protein was extracted from cells and liver tissue using RIPA buffer supplemented with phosphatase and protease inhibitors. Equal amounts of protein from each sample were electrophoresised in 8–12% SDS-PAGE and then transferred to PVDF membranes (Merck, Darmstadt Germany). The membrane was blocked with 5% skimmed milk in tris-buffered saline solution and Tween-20 (TBST) and then incubated overnight in the following primary antibodies: anti-PCYT2 (1:250), anti-PISD (1:500), anti-BAX (1:500), anti-Bcl-2 (1:500), anti-cleaved-caspase3 (1:500), and anti-β-actin (1:5000). Then, the blots were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase. Western blotting was observed using a chemiluminescence solution (Affinity, Changzhou, China) and a protein imager (Bio-Rad).