Il 10

Western blot analysis was performed using routine protocols. Briefly, protein extracts (50 μg) from the whole eye were separated in 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. Rabbit polyclonal antibodies against ICAM-1, IFN-γ, IL-10, cleaved-caspase3 (Wanleibio, China), CD11a, CD18, and TNF-α (Affinity Bioscience, USA) were incubated with the membranes overnight at 4°C. The membranes were washed in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) 3 times, incubated with the indicated HRP-conjugated goat anti-rabbit IgG (Proteintech) antibody for 1 h at room temperature and then washed in TBST 3 times. A Super Signal Chemiluminescent detection system (Bio-Rad) was used to detect the signals.

According to the manufacturer’s instructions, RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract total protein from liver tissue and hepatocytes. Western blotting was performed as previously described (Lei et al., 2016 (link)). The membranes were incubated with primary antibodies against NF-κB/p65 (1:1,000; Proteintech, United States), phospho-NF-κB/p65 (p-p65; 1:1,000; Zen Bioscience, China), TNF-α (1:1,000; Proteintech; United States), IL-6 (1:1,000; Proteintech, United States), IL-10 (1:1,000; Wanleibio, China), Bax (1:2000; Proteintech, United States), Bcl-2 (1:1,000; Proteintech, United States), Nrf2 (1:1,000; Proteintech, United States), HO-1 (1:1,000; Proteintech, United States), β-actin (1:5,000; Proteintech, United States) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; Proteintech, United States). The relative protein expression was analyzed using ImageJ software (NIH, Maryland, United States).