Anti rabbit secondary antibody conjugated to horseradish peroxidase

Roswell Park Memorial Institute (RPMI) 1640 medium, penicillin-streptomycin (Pen Strep), fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Phorbol 12-myristate 13-acetate (PMA), LPS (Escherichia coli 055:B5), RIPA buffer, DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 1× Halt Protease and Phosphatase Inhibitor Cocktail was purchased from Pierce (Rockford, IL, USA). Human TNF-α and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA). Alamar blue reagent for cell viability assay was purchased from Life Technologies (Grand Island, NY, USA). Primary antibodies specific to COX-2, p-p38, p38, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, p-IκBᾳ, IκBᾳ, p-IKKᾳ/β, p-NFκBp65 and β-actin were purchased from Cell Signaling Technology (Beverly, MA) and, in addition, anti-rabbit secondary antibody conjugated to horseradish peroxidase was obtained from Cell Signaling Technology (Beverly, MA). Methanol and acetonitrile of HPLC grade were purchased from Fisher Scientific (Loughborough, UK). Phyllanthin, hypoPhyllanthin, niranthin gallic acid, ellagic acid, corilagin and geraniin (purity > 98%) were purchased from ChromaDex (CA, USA). Dexamethasone was obtained from CCM Duopharma Biotech Bhd (Selangor, Malaysia).

PC12 cells were cultured in 6 well plates with 20 × 10⁴ cells/well for western blotting examination. Immediately following OGD/R, the cells were rinsed with ice-cold PBS and collected. The total proteins were extracted using RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with cocktail protein inhibitors and centrifuged at 12,000 g for 15 min at 4°C. Equal amounts of protein samples (50 μg) were loaded in each lane and proteins were resolved by 8–15% SDS/PAGE. The proteins were then electro-transferred onto a PVDF membrane using a wet transfer system. The membranes were blocked in 5% no-fat milk for 1 h at room temperature and then incubated with primary antibodies (1:1000, Cell Signaling Technology) overnight at 4°C. Following this, the membrane was incubated with anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:2000, Cell Signaling Technology) for 2 h. The membranes were washed and visualized by an enhanced chemiluminescent substrate and scanned with the Kodak Digital Imaging System (5200 Multi, Tanon, China).