Antibody for β actin

Antibodies against HCoV-OC43 N (Mouse anti-HCoV-OC43 N monoclonal Ab, Catalog No. LS-C79764) and S protein (Rabbit anti-HCoV-OC43 S Ab, Catalog No. LS-C371066) were purchased from LifeSpan BioSciences (Seattle, WA, USA). Both the peroxidase-conjugated streptavidin (Catalog No. S5512) and antibody for β-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The antibody against the Fc domain of human IgG (Catalog No. 790-035-098) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Erianin was obtained from ChemFaces Biochemical (Wuhan, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and chloroquine were bought from Sigma-Aldrich (St. Louis, MO, USA). Ad-mCherry-p62 and Ad-mCherry-GFP-LC3B were purchased from Beyotime (Nantong, China). Apoptosis detection kit and propidium iodide (PI)/RNase staining buffer were purchased from BD Pharmingen (San Diego, CA, United States). Antibodies for Cyto C, caspase 9, cleaved caspase 3, GSDMD, CDC25C, CDC2, phospho-CDC2 (p-CDC2, Thr161), cyclin B1 and phospho-4EBP1 (p-4EBP1) were obtained from Cell Signaling Technology (Danvers, MA, United States); antibodies for PARP, NLRP3, 4EBP1, mTOR, phospho-mTOR (p-mTOR), RHEB, LC3B, beclin-1 and p62 were obtained from Proteintech Group (Wuhan, China); antibodies for cleaved-caspase 1, GSDME and PPT1 were from Abcam (Cambridge, MA, United States); and the antibody for β-actin was from Sigma-Aldrich. The secondary antibodies were purchased from Zhong Shan Golden Bridge Biotech Co. (Beijing, China).

Rabbit monoclonal antibody against phospho VEGFR-2 purchased from Cell Signaling, MA, USA was used to detect VEGFR-2 phosphorylation. The protein loads were normalized using antibody for β actin (Sigma, MO, USA) and the antibody reactive bands were quantified by densitometry7 (link)20 (link)21 (link).

NSC-743380 was synthesized as previously described [22 (link)]. Antibody against SULT1A1 was obtained from R&D Systems and antibody for β-actin from Sigma. The pEGFP-N1 plasmid for expressing green fluorescent protein (GFP) was obtained from Clontech. The plasmids expressing constitutively active STAT3 (STAT3CA) and AKT1 (AKT1CA) were described previously [20 (link), 59 (link)]. All other plasmids expressing cDNAs or short hairpin RNA (shRNA) were obtained from either Origene Technologies or Open Biosystems. All plasmids expressing cDNAs were verified by DNA sequencing performed at the Sanger DNA Sequencing Core Facility at our institution.

Dissected hippocampi were immediately immersed individually in a lysis buffer and sonicated for 10 s (n = 3 mice per group). Sodium dodecyl sulfate (SDS) sample buffer (4×) was added to each homogenized sample, and the samples were heated at 100 °C for 10 min. Immunoblotting was performed as described previously [55 (link)]. Briefly, the resolved proteins were separated with SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred onto membrane by using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was incubated with rabbit anti-Nrf2 (1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After extensive washing and incubation with secondary antibodies for 2 h at room temperature, signals were developed using a chemiluminescence kit (SuperSignal^® West Pico PLUS; Thermo Fisher Scientific) and read on a ChemiDoc MP Imaging System (Bio-Rad). To quantify Nrf2 expression, the membrane was re-probed with an antibody for β-actin (1:10,000; Sigma-Aldrich). Several exposure times were used to obtain signals within the linear range, and the bands were quantified using the ImageJ software (NIH, Bethesda, MD, USA).