Grp78

Total protein concentration was measured using the BCA method, and 50 μg protein per lane was separated by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) on 8% or 10% polyacrylamide gels and transferred to poly vinylidene fluoride (PVDF) membranes. Membranes were incubated with primary antibodies for either 2 h at room temperature or overnight at 4°C. The following antibodies were used (at a 1:1,000 dilution unless otherwise indicated): total eIF2α (#5324; Cell Signaling Technology (CST), Danvers, MA, USA), phospho-eIF2α (#3398; CST), total PERK (#20582-1-AP; Proteintech Group, Chicago, IL, USA), phospho-PERK (sc-32577: Santa Cruz, Dallas, TX, USA), total JNK (#9258; CST), phospho-JNK (#4668; CST), GRP78 (#3216-1; Epitomics, Burlingame, CA, USA), ORP150 (#3905-1; Epitomics), CHOP (sc-575; Santa Cruz) and cleaved caspase-3 (#9661; CST). Equal loading of protein between lanes was confirmed by subsequent immunoblotting of β-actin (1:5,000; Sigma, St Louis, MO, USA). After incubation with horseradish peroxidase-conjugated goat anti-mouse or donkey anti-rabbit antibody (1:5,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature, immunodetection was performed by chemiluminescence.

Bovine serum albumin (BSA), 2',7’-Dichlorodihydrofluorescein diacetate (DCFH-DA), resveratrol, and DL-Glyceraldehyde were purchased from Sigma (St. Louis, MO, USA). DMEM/F12 media, fetal bovine serum (FBS), penicillin, streptomycin, and Hanks Balanced Salt Solution (HBSS) were purchased from Invitrogen (Carlsbad, CA, USA). APP 22C11 antibody was purchased from Chemicon (Temecula, CA, USA). Sirt1, NQO-1, and GRP78 antibodies were purchased from Epitomics (CA, USA). APP, Actin antibodies, and enhanced chemiluminescence (ECL) kit were purchased from Millipore (Billerica, MA, USA); and p53, Nrf-2, Ho-1, bcl-2, bax, caspase3, and β-amyloid antibodies were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). Nitrocellulose membranes were purchased from PALL corp. (Ann Arbor, MI, USA).

Cells were lysed in lysis buffer (Beyotime, China) mixed with 1% protease inhibitors (Beyotime, China) to extract total protein. Mitochondrial fractions were extracted using a Cell Mitochondria Isolation Kit (Beyotime, China). The cell medium was centrifuged (800 × g, 5 min), and the supernatant was filtered for the detection of high mobility group box 1 protein (HMGB1), as described previously [17 (link)]. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Total cell lysates were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Amersham Biosciences, USA). Blots were probed with the primary antibodies against phosphorylated PKR-like ER kinase (p-PERK, 1 : 1000; CST, USA), GRP78 (1 : 1000; Abcam, UK), CHOP (1 : 1000; Abcam, UK), HMGB1 (1 : 10000; Abcam, UK), PARP (1 : 500; Abcam, UK), VDAC1 (1 : 1000; Abcam, UK), or GAPDH (1 : 1000; Tianjin Sun gene Biotech Co, China) and incubated (with rocking) at 4°C overnight. Membranes were probed with secondary antibodies and visualized using an enhanced chemiluminescence reagent (ECL, Amersham Biosciences, Piscataway, NJ, USA).