Keap1 sc 514914

Cells were inoculated into a 6-well plate and divided into normal, Al(mal)₃ treatment, Andro treatment and Al(mal)₃+Andro co-treatment groups. Total proteins were extracted by RIPA lysis buffer and quantified by BCA protein assay kit (ThermoFisher, USA). Each sample (20 μg) was separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were then blocked with 5% (w/v) fat-free milk for 1 h, followed by incubation overnight at 4 °C with primary antibodies at 1:1000 dilution ratio: APP(1007-5, huaanbio, China), BACE1(5606, CST, USA), p-Tau (Ser396) (AF3148, Affinity, USA), Tau (AF1249, Beyotime, Shanghai, China), Keap1 (sc-514914, Santa Cruz, USA) Nrf2 (ab62352, Abcam, USA), p62 (AF5384, Affinity, USA), LC3 (bs-8878R, Bioss, China), GAPDH (FD0063, Fudebio, China). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The membranes were rinsed in the western lighting plus-ECL solutions, and finally the immunoreactive bands were detected using Chemiluminescence Imaging System (ChemiScope 3000 Mini, Clinx, China). The relative optical density of the digitized image was analysed by Image J software.

Paeoniflorin (purity: 98.04%) was acquired from MedChemExpress (Monmouth Junction, NJ, United States). Tin protoporphyrin IX (SnPP) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Primary antibodies were used as following: C-caspase3 (ab32351), Bax (ab32503), Bcl-2 (ab182858), and Cytochrome C (ab133504) were obtained from Abcam (Cambridge, United Kingdom); Keap1 (sc-514914) and poly (ADP-ribose) polymerase (PARP) (sc-7150) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibodies against Nrf2 (16396-1-AP), HO-1 (27282-1-AP), Lamin B (12987-1-AP), NF-κB (66535-1-Ig) and β-actin (66009-1-Ig) were acquired from Proteintech Group (Chicago, IL, United States), and 4′, 6-diamidino-2-phenylindole (DAPI) was received from Beyotime (Shanghai, China). ML385 was purchased from ChemeGen (Shanghai, China). All experimental consumables for cell culture are from Gibco (Grand Island, NY, United States).