Goat anti rabbit horseradish peroxidase labeled antibody

Protein expression levels of 3-nitrotyrosine and endothelial nitric oxide synthase (eNOS) were examined by western blot assay. Total protein was extracted from the corpus cavernous tissue using mammalian protein extraction reagent (Pierce, Rockford, IL, USA) and quantified using the bicinchoninic acid protein assay kit (Pierce). The extracted total proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinyl difluoride membranes. After blocking with 5% fat-free milk for 30 minutes, the membranes were incubated overnight with rabbit anti-rat 3-nitrotyrosine (ab52309; 1:1,000), eNOS (ab5589; 1:1,000), phospho-eNOS (ab51038; 1:500), and β-actin (ab8227; 1:1,000) antibodies (Abcam, Cambridge, UK) at 4°C for 24 hours. The secondary goat anti-rabbit horseradish peroxidase-labeled antibody (1:2,000; Abcam) was added and incubated at 4°C for 1 hour. Finally, the protein bands were visualized using an enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA). The gray values were measured using an ultraviolet photometry imaging system (UVP LLC, Upland, CA, USA). The housekeeping gene β-actin was used as an internal control for normalization to determine protein expression levels.