Enhanced chemiluminescence ecl system

Western blotting assay was used to detect the protein expression of endothelial nitric oxide synthase (eNOS) in the corpus cavernosum. Rat penile tissues were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and the supernatant was collected after centrifugation at 12 000 g for 15 min at 4 °C. The protein concentration was determined by using the BCA protein assay reagent kit (Beyotime Institute of Biotechnology). Proteins were fractionated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes, which were blocked with 5% milk at room temperature for 1 h and incubated with rabbit eNOS antibody (Cell Signaling Technology, Inc, Boston, MA, USA) overnight. After washing in Tris-buffered saline with 0.1% tween 20 (TBST) three times for 10 min, the membrane was incubated for 1 h in darkness with horseradish peroxidase (HRP)-conjugated rabbit antibody (Boster, Wuhan, China). The protein bands were detected by using the enhanced chemiluminescence (ECL) system (Beyotime Institute of Biotechnology) followed by autoradiography. Results were quantified by densitometry, and GAPDH was used as an internal reference to normalize the data.

Total proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) buffer and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Following the transfer of separated proteins onto a polyvinylidene difluoride membrane, the membrane was blocked with 5% nonfat milk in Trisbuffered saline with Tween 20 (TBST; 10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at room temperature for 1 h, washed three times in TBST, and incubated with primary antibodies against SIRT1 (Abcam, Cambridge, MA, USA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. After three washes with TBST, the membrane was incubated with horseradish peroxidaseconjugated secondary antibodies at room temperature for 2 h. The signal was developed using the enhanced chemiluminescence (ECL) system (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer's instructions.