Ecl plus reagent kit

On day 7 after surgery, tissues (1 cm × 1 cm) from Area II were dissected and stored at −80 °C prior to Western blotting. Protein concentrations were determined using the BCA assay (Thermo, Rockford, IL, USA). Seventy microgram amounts of protein were separated on a 12% (w/v) gel and transferred onto PVDF membranes (Roche Applied Science, Indianapolis, IN). After blocking with 5% (w/v) non-fat milk for 2 h, the membranes were incubated with antibodies against VEGF (1:400; Bioworld, Nanjing, China), IL1β, IL6, GAPDH (1:1000; Abcam), Beclin1, p62, LC3 (1:1000; Cell Signaling Technology), and β-actin (1:200; Santa Cruz Biotechnology). Next, the membranes were incubated with a goat-anti-rabbit secondary antibody for 2 h at room temperature and bands detected using the ECL-plus reagent kit (PerkinElmer, Waltham, MA, USA). Band intensity was quantified using the Image Lab 3.0 software (Bio-Rad).

NBP (C_₁₂H_₁₄O_₂; purity, 99.98%), rapamycin, H&E staining kit, DAB developer, and pentobarbital sodium were provided by Solarbio Science & Technology (Beijing, China). The primary antibody against CDH5 was acquired from Boster Biological Technology (A02632-2; Hangzhou, China). GAPDH, SOD1, MMP9, HO1, CTSD, and CASP3 were acquired from Proteintech Group (60004-1-Ig, 10269-1-AP, 12452-1-AP, 10375-2-AP, 10701-1-AP, 21327-1-AP, and 19677-1-AP; Chicago, United States). Antibodies for p62, CYC, Bax, eNOS, and LC3 were purchased from Cell Signaling Technology (5114T 4272T, 14796, 32027, and 3868; Beverly, MA, United States). HRP-conjugated IgG secondary antibody and FITC-conjugated IgG secondary antibodies were obtained from Affinity Biosciences (s0001, s0002; Jiangsu, China). Other reagents include DAPI solution (Beyotime Biotechnology, Jiangsu, China, a BCA Kit (Thermo Fisher Scientific, Rockford, IL, United States) and an ECL Plus Reagent Kit (PerkinElmer Life Sciences, Waltham, MA, United States) which were used according to manufacturer’s instructions.