Anti enos antibody

Fresh penile tissue was pulverized and added into the immunoprecipitation lysis solution containing specific enzyme inhibitors (Beyotime Biotechnology) to extract the protein. The bicinchoninic acid protein concentration measurement kit (Beyotime Biotechnology) was used to quantify the protein content of supernatants after centrifugation. The supernatant was incubated for 1 hour at 4 °C with rabbit common IgG (Santa Cruz) and protein A + G agarose (Santa Cruz). After centrifugation, anti-eNOS antibody (Abcam) and resuspended agarose (Santa Cruz) were added to the supernatant, and the whole thing was incubated at 4 °C overnight. Then, the sample was centrifuged for 5 minutes to collect the immunoprecipitates. The immunoprecipitates were washed 5 times with the immunoprecipitation lysis solution, each time repeating the centrifugation step. After the final wash, the supernatant was removed; loading buffer was added; and the protein was denatured by boiling and stored at −20 °C. The remaining procedures were the same as those used for Western blotting.

To extract the protein, the penile spongy tissue was pulverized and added into the radioimmunoprecipitation assay lysis buffer containing enzyme inhibitors (Beyotime Biotechnology). The bicinchoninic acid protein concentration measurement kit (Beyotime Biotechnology) was used to quantify the protein content of supernatants after centrifugation. The supernatant was mixed with a loading buffer and heated to 100 °C to denature the protein. The samples were stored at −20 °C. After electrophoresis, transmembrane, and membrane closure, the polyvinylidene difluoride membranes were incubated with primary antibodies at 4 °C overnight: anti-eNOS antibody (1:1000; Abcam), anti-phospho-eNOS-Ser¹¹⁷⁷ antibody (1:1000; Abcam), and anti-GIT1 antibody (1:100; Santa Cruz Biotechnology). After the membranes were rinsed, the horseradish peroxidase–conjugated secondary antibody (1:1000; Proteintech) was added and incubated at room temperature for 1 hour. The protein strips were presented by the protein imager (Bio-Rad Laboratories) after addition of the enhanced chemiluminescence solution. The grayscale value of the protein strips was quantitated with QuantityOne 4.6 software (Bio-Rad Laboratories).¹⁹ (link)^,22 (link)

Celiac artery tissue was fixed in 10% formalin neutral buffer solution and paraffin-embedded into tissue blocks. Serial 3-μm-thick sections were cut and placed on glass slides for immunohistochemical staining using an EnVision^TM detection system (DakoCytomation A/S, Glostrup, Denmark) according to the manufacturer’s recommendations. Endogenous peroxidases in the specimens were blocked with Peroxidase-Blocking Solution (DakoCytomation A/S) for 5 min at room temperature, and after incubation with Protein Block Serum-Free reagent (DakoCytomation A/S), an anti-eNOS antibody (prediluted; Abcam, Tokyo, Japan) was applied as the primary antibody. Following incubation for 90 min at room temperature and washing, the tissues were incubated with EnVision^TM/HRP Rabbit/Mouse secondary antibodies (DakoCytomation A/S) for 30 min at room temperature and then with the chromogen 3,3′-diaminobenzidine. Nuclei were counterstained using Mayer’s hematoxylin (Wako Pure Chemical Industries Ltd.). Tissue sections that were not treated with primary antibody were used as negative controls. The staining intensities of the tissue samples were determined semi-quantitatively (none, 0; weak, 1; moderate, 2; intense, 3).

Proteins of eNOS, PGI₂ synthase (PGIS), IP, and β-actin (internal controls) in abdominal aortas were detected by Western blots. Briefly, vessels were minced in ice-cold RIPA buffer containing proteinase inhibitor cocktail (Roche Applied Science, Mannheim, Germany), followed by homogenizing using a glass homogenizer. Total proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane, which was then sequentially probed with respective first and secondary antibodies. Anti-eNOS antibody (rabbit polyclonal; 1:1,000 dilution) was purchased from Abcam (Cambridge, MA, United States), while anti-PGIS (rabbit polyclonal; 1:2,000 dilution) and anti-IP (rabbit polyclonal; 1:2,000 dilution) antibodies were bought from Cayman Chemical (Ann Arbor, MI, United States). Anti-β-actin (rabbit polyclonal; 1:5,000 dilution) antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Immunocomplexes were visualized with SuperSignal West Femto maximum sensitivity substrate (Pierce, Rockford, IL, United States) and detected by ChemiDoc XRS+ chemiluminescence imager (Bio-Rad, Hercules, CA, United States). Band densities, which were analyzed by Image Lab software version 4.1 (Bio-Rad), were expressed relative to β-actin and normalized by the average value of controls.

To investigate the effects of EMPs on relevant protein expression in mouse hearts, isolated mouse hearts treated without or with EMPs were embedded with paraffin, sectioned and subjected to immunostaining. The expression of caveolin-1, eNOS, and P38 MAPK in mouse hearts was detected by immunohistochemical staining using standard protocols. Polycolonal rabbit IgG anti-Caveolin-1 antibody (1:100, Abcam), anti-eNOS antibody (1:100, Abcam) and anti-p38 MAPK antibody (1:100, Abcam) were used as primary antibodies.

eNOS protein expression was assessed by immunohistochemical staining using the Bond Polymer Intense Detection System (Vision Biosystems, Mount Waverley, Victoria, Australia) according to the manufacturer's instructions. To summarize, 4-μm sections of formalin-fixed, paraffin-embedded tissue were deparaffinized with Bond Dewax Solution (Vision BioSystems), and antigen retrieval was performed using Bond ER Solution (Vision BioSystems) for 30 min at 100°C. Endogenous peroxidases were quenched by incubating sections in hydrogen peroxide for 5 min. Sections were then incubated for 15 min at ambient temperature with a rabbit polyclonal anti-eNOS antibody (1:100; Abcam, Cambridge, MA, USA). The biotin-free polymeric horseradish peroxidase-linker antibody-conjugate system was used in the Bond-maX automatic slide stainer (Vision BioSystems), and visualization was performed using a 3.3-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris-HCl buffer [pH 7.6], and 0.006% H₂O₂). Nuclei were counterstained with hematoxylin. Slides were subsequently dehydrated following a standard procedure and sealed with coverslips. To minimize inter-assay variation, positive and negative control samples were included in each run. The positive control sample was normal liver tissue, while the negative control was prepared by substituting non-immune serum for the primary antibody. No staining was detected.