Ddah1

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).

Myocardial tissue and cardiomyocytes were lysed with RIPA buffer for the determination of protein arginine N-methyltransferase 1 (PRMT1), dimethylarginine dimethylaminohydrolase1 (DDAH1) or DDAH2, eNOS, PGC-1α, UCP2 and β-actin (the former three antibodies were products of Abcam, Cambridge, MA, USA from rabbit, goat, goat, respectively; and the latter four antibodies from Santa Cruz Biotechnology, Santa Cruz, CA, USA from rabbit, rabbit, goat, rabbit, respectively) expression by Western blotting as described previously [25 (link)].
Immunoprecipitation was employed to detect post-translational acetylation and phosphorylation of PGC-1α as described by Lei S et al [28 (link)]. In this experiment, a total of 400 μl cell lysates were subjected to immunoprecipitation with 1 μg PGC-1α primary antibody at 4 °C overnight. The antibody-bound proteins were precipitated with 15 μl protein G magnetic beads. After separating from protein G magnetic beads, immunoprecipitates were subjected to Western blotting as described above with the antibody against acetylated-lysine or phosphorylated-serine/threonine (protein G magnetic beads and antibodies were products of Cell signaling technology, Danvers, MA, USA, the latter from rabbit), respectively.

Total protein samples were extracted from each sample after treatment as described in [29 (link)] and measured using a Bicinchoninic acid (BCA) kit (Beyontime, Jiangsu, China). Lysates (20 μg each sample) were separated by 10% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk in TBST for 90 min at room temperature and subsequently incubated with corresponding antibodies as follows: DDAH1 (1 : 1000, Abcam, USA), p-eNOS (ser1177) (1 : 1000, Cell Signaling Technology, USA) and GAPDH (1 : 5000, Sigma, USA) at 4°C overnight. After incubation, membranes were washed three times with TBST and then incubated with HRP-conjugated secondary goat-anti-rabbit IgG antibody for 1 h at room temperature. The signal was detected and ratio of the DDAH1 against GAPDH control was calculated using the Bio-Rad Chemi Doc XRS+ Imaging System (Bio-Rad Biosciences, USA).