Hepatic Antioxidant Protein Extraction and Analysis

Total protein from hepatic tissue was extracted with a commercial kit (Beyotime Biotechnology, Jiangsu, China) and homogenized according to the manufacturer's protocol. Protein content was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, U.S.A.). The antibodies used in this study were as follows: anti-GSH-Px1 (1 : 2000, Abcam), anti-CAT (1 : 2000, Protein Tech), anti-Nrf2 (1 : 2000, Protein Tech), anti-SOD2 (1 : 5000, Novus), anti-NQO1 (1 : 1000, Abcam), anti-HO-1 (1 : 1000, Abcam), anti-eNOS (1 : 1000, CELL SIGNALING), anti-iNOS (1 : 1000, Cell Signaling), and anti-β-actin (1 : 1500, Santa Cruz). Following 1 h of incubation by the HRP-conjugated goat anti-rabbit IgG secondary antibody, signals were detected using the enhanced chemiluminescence kits (ECL-Plus, Thermo, Waltham, MA, USA); afterwards, they were screened to detect the fluorescence using the BioRad gel detection system. The densitometric values were standardized based on β-actin, and expressed relative to control.^{32 (link)}