Af0096

Immunohistochemistry was performed as previously reported [37 (link)]. After antigen retrieval and dewaxed, sections of corpus cavernosum were incubated with antibodies against endothelial nitric oxide synthase (eNOS; 1:100; Affinity Biosciences, AF0096, USA) or neuronal nitric oxide synthase (nNOS; 1:100; Affinity Biosciences, AF6249, USA) antibodies, followed by biotinylated secondary antibodies.
The tissues were fixed for 24 h in 4% paraformaldehyde (Servicebio, China) before being imbedded in paraffin. Serial 4 μm paraffin-embedded slices were obtained and dewaxed in xylene I and xylene II for 10 min each, before being rehydrated in a variety of ethanol concentrations (100% for 5 min, 100% for 5 min, 95% 5 min, 90% 5 min, 80% 5 min, 70% 5 min). The slices were then washed three times in distilled water (5 min each). Finally, according to the manufacturer's instructions, sections were stained with H&E solution (Servicebio, China).
Masson’s trichrome staining was conducted according to the standard protocol. The area of smooth muscle and collagen was determined with ImageJ software. The ratio of smooth muscle to collagen was taken as an indicator of the degree of fibrosis in the corpus cavernosum.

Total protein was extracted from the frozen thoracic aorta of rats and HUVECs with RIPA lysis buffer containing protease inhibitor cocktail. The protein concentrations were determined by the Bradford protein assay kit. Equal amounts of protein lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes followed by a block with 5% skimmed milk at 4°C for 1.5 h. Subsequently, the membranes were incubated with primary antibodies against eNOS (AF0096, Affinity, 1:1,000) and p-eNOS (AF3247,Affinity, 1:1,000), c-jun N-terminal kinase (JNK BS1544, Bioworld, 1:500) and p-JNK (BS4322, Bioworld, 1:500), B-cell lymphoma-2 (Bcl-2, BA0412, Boster, 1:800), BCL2-Associated X (Bax, BA0315-2, Boster, 1:800), inositol-requiring enzyme 1α (IRE1α, bs-8680R, Bioss, 1:1,000), glucose regulated protein 78 (GRP78, ab21685, Abcam, 1:1,000), CHOP, respectively. β-actin (M02014-5, Boster, 1:1,500) was used as the internal reference. Antibody binding was detected with enhanced chemiluminescent agent and quantified with Image J software. Each experiment was repeated three times.

Tissue protein samples were prepared in RIPA buffer (Beyotime Biotechnology, Haimen, China) containing phosphatase inhibitor and protease inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA). Samples containing 20 μg of protein were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with Tris‐buffered saline‐Tween with 5% bovine serum albumin and incubated at 4°C overnight with primary antibodies against α‐SMA (1:1000; AF1032, Affinity Biosciences), Collagen I (1:1000; AF7001, Affinity Biosciences), TGF‐β1 (1:1000; AF1027, Affinity Biosciences), eNOS (1:1000; AF0096, Affinity Biosciences), nNOS (1:1000; AF6249, Affinity Biosciences) or β‐actin (1:1000; 20536‐1‐AP, Proteintech, China). After hybridization of secondary antibodies (1:5000 or 1:10000; ProMab, USA), the immunoreactive proteins were detected through an enhanced chemiluminescence detection system (Pierce, Thermo Fisher Scientific, Rockford, IL, USA).