Nrf2 phospho s40

Mep-S was synthesized in our laboratory as previously described [19 (link)]. Scopolamine, H₂O₂, and N-acetylcysteine (NAC) were the products of Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM)/F-12 (1 : 1), phosphate-buffered saline (PBS), and a mixture of penicillin and streptomycin were the products of Gibco (Grand Island, NY, USA). The rabbit-derived antibodies against NAD(P)H quinine oxidoreductase-1 (NQO-1; cat #ab80588), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein-1 (Keap1), Nrf2, phospho-Nrf2 (S40), and B-cell lymphoma 2 (Bcl-2) were the products of Abcam (Cambridge, MA, USA). The rabbit-derived antibodies against caspase-3 (D3R6Y), phospho-Akt (Ser473), Akt, and Bax were the products of Cell Signaling Technology (CST; Danvers, MA, USA). Mep-S was dissolved in dimethyl sulfoxide (DMSO; Sigma), and NAC was dissolved in normal saline to obtain stock solutions (20 mg/ml), which were diluted with saline (for in vivo experiments) or culture medium (for in vitro tests). The Mep-S working solutions contain less than 2.5% of DMSO.

Protein expression was analyzed using a slightly modified method, as previously reported by [32 (link)]. The cells were lysed with RIPA buffer containing 1% PMSF and phosphatase inhibitor cocktail (Bioworld, Shanghai, China), scraped on ice, and incubated at room temperature for 60 min. The homogenate was centrifuged at 13000 rpm for 18 min, and the protein concentration of the supernatant was determined using the BCA protein detection kit (Beyotime, Shanghai, China). Equal amounts of protein were separated using 10% SDS-PAGE and transferred to a nitrocellulose membrane (Pall, New York, USA). Nonspecific binding to the membrane was blocked in 5% skimmed milk, and the membranes were incubated with antibodies to β-actin (1 : 5000 dilution; Promega Corporation, USA), Nrf2, Keap1, HO-1, NQO1, Bax, Bcl-2 (1 : 2500; Proteintech, USA), and Nrf2 (phospho S40) (1 : 5000; Abcam, United Kingdom). The membranes were incubated overnight at 4°C and incubated with 1 : 10000 mouse/rabbit secondary antibody for 1 h. The immune response was detected by ECL Plus™ Western Blotting Detection System (Pierce, Rockford, USA) and imaged using a gel imaging system (ChemiDoc™ XRS+, Bio-Rad). Finally, ImageJ software was used to calculate the gray value of each protein band to determine the relative expression of the target protein compared with the control protein.