P enos

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).

Blocks of paraffin-embedded aorta tissue were sectioned to a thickness of 7 µm, placed on a coating slide and dried at 40 °C for 24 hrs. Slides were deparaffinized, treated with normal animal serum to block non-specific binding, incubated with antibodies (ET-1 (abcam; UK, dilution rate 1:200), peNOS (abcam; dilution rate 1:250), UCP-1 (Santa Cruz Biotechnology, Inc.; dilution rate 1:200), Sirt1 (Santa Cruz Biotechnology, Inc.; dilution rate 1:200)) for two days at 4 °C, and then rinsed three times with PBS. Slides were then incubated for 1 h with Alexa Fluor Plus secondary antibodies (Thermo Fisher Scientific, MA, USA, dilution rate 1:500) and rinsed three times with PBS. Next, slides were incubated for 5 min with 4′ 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) solution and then rinsed three times with PBS. Finally, cover slips and shield solution were added (Vector Laboratories) followed by detection of fluorescent signals using a confocal laser microscope (LSM 710; Carl Zeiss, Germany). Fluorescent intensity was measured by Zen software 2012 version (Carl Zeiss) and three random samples were taken per sample and measured using the Zen software.

Total EPCs protein were extracted and quantified by protein extraction reagent (Merck) and bicinchoninic acid protein assay kit (Thermo Fisher) separately. Protein extracts were subjected to SDS‐PAGE, transferred to polyvinylidene fluoride membranes (Roche). The following antibodies were used: rabbit anti‐CXCR4 antibody (1:500; ABCAM, USA), rabbit anti‐actin antibody (1:2000; Cell Signaling Technology), rabbit anti‐VEGFa antibody rabbit (1:500; Santa Cruz, USA), rabbit anti pan protein kinase B (1:1000, Abcam), p‐Akt (1:2000, Ser473; Abcam), rabbit anti human PDGF B (1:2000, Abcam), goat anti human FGF 23 (1:800, Abcam), mouse anti human eNOS (1:500 Abcam), p‐eNOS (1:500 Ser1177; Abcam), and rabbit anti‐GADPH antibody (1:3000; Cell Signaling Technology). Proteins were visualized with HRP‐conjugated anti‐rabbit or anti goat IgG (1:2000; Cell Signaling Technology), followed by use of the ECL chemiluminescence system (Thermo).