Clarity western chemiluminescent ecl substrate kit

Polyphenolic compounds, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) disodium salt (ABTS), fluorometric intracellular ROS kit, penicillin/streptomycin, and 2-(4-amidinophenyl)-6-indolecarbami dine dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Parishin derivatives were bought from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China). Fetal bovine serum (FBS) was obtained from Young in Frontier Company (Seoul, Korea). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was bought from MedChemExpress (Princeton, NJ, USA). Ham’s F-12 Nutrient Mix medium, BCA protein assay kit, and cDNA synthesis kit (ReverTra Ace qPCR RT Kit) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Eagle’s Minimum Essential Medium (EMEM) was obtained from ATCC (Manassas, VA, USA). H₂O₂ was purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). The SYBR green qPCR Kit was supplied by TOYOBO (Osaka, Japan). Lactate dehydrogenase (LDH) cytotoxicity assay kit was bought from TAKARA (Shiga Prefecture, Japan). Clarity western chemiluminescent (ECL) substrate kit was supplied by Bio-Rad Laboratories (Hercules, CA, USA).

Hippocampus and SH-SY5Y cells were lysed by tissue protein extraction reagent (T-PER) (Thermo Scientific, Waltham, MA, USA), or RIPA buffer (Biosesang, Gyeonggi-do, South Korea). After centrifugation (13000× g, 4 °C, 15 min), total protein concentration in the supernatant was measured with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). Protein was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was incubated with 5% non-fat milk (Bio-Rad Laboratories, California, USA) in Tris-buffered saline with Tween-20 (TBST) at 22 °C for 2 h. Primary antibodies were diluted according to the manufacturers’ specifications (Table 1) and incubated at 4 °C overnight. The membrane was washed with TBST and then incubated with secondary antibodies (goat anti-rabbit IgG-HRP) (Thermo Scientific, Waltham, MA, USA) for 2 h. Bands were detected using a clarity western chemiluminescent (ECL) substrate kit (Bio-Rad Laboratories, Hercules, California, USA), and band images were obtained using a LAS 500 image system (GE Healthcare, UK). The bands were quantified with Quantity One software (Bio-Rad Laboratories, Hercules, California, USA).