Hmox1

Western blot analysis was performed using a previously reported method^{63 (link)}. In brief, cytoplasmic and nuclear extracts were prepared using a Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. Equal amounts of protein, as measured using a BCA protein assay kit, were mixed with 6 × SDS reducing sample buffer and boiled for 10 minutes before loading. The proteins (50 µg/lane) were then separated on an SDS polyacrylamide gel and transferred electronically to PVDF membranes. After the membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature, they were incubated with primary antibodies against MDA (1:200), 4-HNE (1:200), aconitase-2 (1:200), HMOX1 (1:200), COX2 (1:200), SOD1 (1:200), GPX1 (1:200), p38 (1:200) or p-p38 (1:200) (both from Abcam, Cambridge, CA), or β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. After three washes with Tris-buffered saline with 0.05% Tween 20 for 10 minutes each, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:1000) or goat anti-rabbit IgG (1:1000) for 1 hour at room temperature. The signals were then detected with a chemiluminescence reagent (ECL; GE Healthcare), and the images were acquired using an imaging station (model 2000R; Eastman Kodak, Rochester, NY).

Astragaloside IV (> 98%) was purchased from Nanjing Spring & Autumn Biological Engineering (Nanjing, China) and stored in the dark at −20°C and then dissolved in dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China). It was also diluted with complete medium prior to experimentation. H₂O₂ and t-BHQ were obtained from Sigma-Aldrich (St. Louis, MO, USA). Penicillin and streptomycin, Fetal bovine serum (FBS), and Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). Primary antibodies used targeted NFE2L2 (16396-1-AP, Proteintech, Chicago, IL, USA), NQO1 (11451-1-AP, Proteintech, Chicago, IL, USA), HMOX1 (ab13248, Abcam, Cambridge, UK), and β-actin (GB12001, Servicebio, Wuhan, China). GSH-Px, SOD, CAT, T-AOC, and MDA test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).