eNOS Expression Test: Western Blot Analysis

Corpora tissue was homogenised in ice-cold lysis buffer containing 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 μM leupeptin, 20 μg/ml chymostatin, and 2 mM phenylmethanesulphonyl fluoride (PMSF). Following centrifugation at 12,000 g for 20 min at 4 °C, the supernatant was extracted and quantified using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to membranes. The membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 and then probed with anti-eNOS antibody (1:1000; ab5589, Abcam, Cambridge, UK), anti-phosphorylated-eNOS (P-eNOS) antibody (1:1000; #9571, Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (1:10000; SC47778, Santa Cruz Biotechnology, Dallas, TX, USA) as an internal control. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Densitometric analysis of band intensity was detected by Luminescent Image Analysis System (LAS-3000; Fujifilm, Tokyo, Japan) and measured using Multi Gauge 3.0 software (Fujifilm) [29].