Frozen penile tissues were isolated and prepared in the RIPA buffer containing a protease inhibitor cocktail and sodium fluoride, followed by centrifugation at 12,000 × g for 10 min at 4°C,as described in our previous studies [24 (link)]. Equal amounts (40μg/lane) of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Immobilon-P Transferred Membrane; Millipore Corporation, Billerica, MA, USA). After blocking in 5% bovine serum albumin for 1 h at room temperature, the membranes were incubated with antibodies against: hKLK1 (1:5000; Sigma Aldrich, St. Louis, MO, USA), rKLK1 (1:1000; Sigma Aldrich), COX-2 (1:500; Abcam, Cambridge, MA, USA), PTGIS (1:1000; Abcam), DDAH1 (1:1000; Abcam), DDAH2 (1:1000; Proteintech, Wuhan, Hubei, China), eNOS (1:1000; Abcam), P-eNOS (T495; 1:1000; Abcam), P-eNOS (S1177; 1:500; Abcam), nNOS (1:1000; Abcam) and β-actin (1:1000; Proteintech) overnight at 4°C.
After washing three times in TBST for 30 min, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Proteintech) for 1 h followed by a further 30 min washing, Finally, the bands were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Rockford, IL, USA).
After washing three times in TBST for 30 min, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Proteintech) for 1 h followed by a further 30 min washing, Finally, the bands were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Rockford, IL, USA).