Anti nnos

The protein levels in the penile tissues were measured by Western blotting. After the penis tissue was taken out, it was fully ground in liquid nitrogen. It was then lysed with radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific), and the protein concentrations were detected by a bicinchoninic acid protein detection kit. In 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, equal amounts of protein were injected, electrophoresed, and transferred to polyvinylidene fluoride membranes. These membranes were blocked with 5% skimmed milk for 2 hours and incubated overnight at 4 °C with the following primary antibodies: anti–neuronal nitric oxide synthase (anti-nNOS, 1:1000; Abcam), anti-GRP78 (1:1000; Abcam), anti-CHOP (1:1000; CST), anti-PERK (1:1000; CST), anti–p-PERK (1:1000; CST), anti–caspase 3 (1:1000; Novus Biologicals), anti–α-SMA (1:1000; CST), and anti-OPN (1:1000; Novus Biologicals). After the membranes were washed 3 times, they were incubated with secondary antibody at room temperature for 1 hour. An enhanced chemiluminescence system (SH-Compact 523; SHST) was used to detect the protein bands, which were then quantitatively analyzed with ImageJ (version 1.52a; National Institutes of Health). The protein levels were normalized to the levels of β-actin or GAPDH (1:1000; CST).

After the animals were euthanized, the antrum was collected and stored immediately at −80°C. Total protein was extracted from gastric antrum homogenate, and the concentration of protein was determined using an enzyme-linked immunosorbent assay at 562 nm. The protein sample was boiled, and then equivalent amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 8% or 12% gels. The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. TBS-T buffer (Tris buffer saline, 0.1% Tween) containing 5% BSA was used to block the membrane at room temperature for 60 minutes. Then, the membrane was incubated with the primary antibodies at 4°C overnight. Horseradish peroxidase (HRP) conjugated secondary antibodies were then incubated with the membranes at room temperature for 60 minutes. The gray values of the immunoreactive protein bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). The antibodies used included anti-PGP9.5 (rabbit, 1 : 1000, Abcam, Cambridge, UK), anti-nNOS (rabbit, 1 : 1000, Abcam), anti-ChAT (rabbit 1 : 1000, Abcam), anti-GAPDH (1 : 1000, Cell signaling Technology, Danvers, MA, USA), and anti-Vinculin (1 : 2000, Abcam, Cambridge, UK) antibodies.