Antioxidant Protein Expression Analysis

Western blot analyses were performed as previously described (Wang et al., 2011 (link)). Animals were anesthetized and sacrificed by cervical dislocation. The brain tissue was immediately harvested and stored at −80°C. The tissue dissected from mouse brain was homogenized in RIPA buffer. After centrifuging at 12,000 rpm for 5 min, supernatant was collected as the total cell lysate. Protein concentration was measured using the BCA Protein Assay Kit from Thermo Fisher Scientific. Supernatant was separated on 4%–15% polyacrylamide Tris-HCl gradient gels (BioRad, Hercules, CA, USA) and transferred to PVDF membranes. The integrated optic density of the immunoreactive band was quantified with NIH ImageJ software. The primary antibodies used were: rabbit polyclonal superoxide dismutase 2 (SOD2; 1:2000; EMD Millipore), rabbit polyclonal glutathione peroxidase 1 (GPx1; 1:1000; Abcam, Cambridge, MA, USA), goat polyclonal NAD(P)H:Quinone Oxidoreductase 1 (NQO1; NQP1; 1:1000; Abacam), rabbit polyclonal heme oxygenase 1 (HO1; 1:1000; Enzo Life Sciences, Farmingdale, NY, USA) and mouse anti-actin (1:5000, EMD Millipore).