Pierce ecl western blotting substrate

The protein concentration in the hippocampus or cells was detected using a BCA kit (Meilunbio, Dalian, China). Briefly, the total protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Following the blocking step with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with specific primary antibodies, namely β-actin (1:2000) (Elabscience Biotechnology, Wuhan, China), ALOX15 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Nrf2 (1:500), HO-1 (1:500), VDR (1:1000), GPX4 (1:1000), NeuN (1:1000), and ACSL4 (1:500) (Affinity, Changzhou, China). After washing by TBST, the membranes were incubated with peroxidase-conjugated goat anti-rabat or goat anti-mouse IgG (EarthOx, San Francisco, CA, USA) for 1 h at room temperature. Then, the protein bands were visualized using Pierce ECL Western blotting Substrate (Engreen Biosystem, Beijing, China). The relative optical density values of protein bands were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

The protein expression levels of SOD-1, CAT and GSS were assessed via Western blot analysis. Briefly, total protein (20 μg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane with a Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories, Richmond, Calif.). The membranes were blocked with 5% skim milk powder dissolved in Tris-buffered saline containing 0.1% Tween 20. The membranes were then incubated with SOD-1/CAT/GSS rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C and then with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The protein bands were visualized using Pierce ECL Western Blotting Substrate (Engreen Biosystem, Beijing, China). The relative density of bands was assessed by densitometry using ImageJ software (http://rsbweb.nih.gov/ij/download.html).