2.5. Western Blot Analysis

BV2 microglial cells were treated with amentoflavone (10 μΜ) for 1 h, followed by treatment with LPS (1 μg/ml) for 6 h. The total protein or nuclear protein from the BV2 microglial cells was prepared and extracted using the Membrane and Cytoplasmic Protein Extraction Kit (C510005, Sangon Biotech, Shanghai, China) or Nucleoprotein Extraction Kit (C500009, Sangon Biotech). Protein concentration was measured using the BCA Protein Assay Kit (KGP902, KeyGEN, Jiangsu, China). Equal amounts of protein (50 μg per lane) were resolved on a 12% sodium dodecyl sulfate- (SDS-) polyacrylamide gel (SDS-PAGE) and then transferred onto 0.22 μM polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat milk, rabbit anti-TLR4 (1 : 500), iba-1 (1 : 1000), MyD88 (1 : 2000), p-IκB (1 : 500), IκB (1 : 500), NF-κB p65 (1 : 1000), IL-1β (1 : 500), TNF-α (1 : 1000), iNOS (1 : 1000), Nrf2 (1 : 500), Keap1 (1 : 500), HO-1 (1 : 1000), PCNA (1 : 500), and β-actin (1 : 2000) were incubated overnight at 4°C. Membranes were washed with PBST (PBS containing 1‰ Tween 20) and subsequently incubated with peroxidase-conjugated goat antirabbit IgG (H + L) (1 : 5000). Detection was performed with the automatic chemiluminescence gel imaging analysis system (Amersham Imager 600, General Electric Company, USA) and quantified via densitometry with ImageJ software. All experiments were performed in triplicate.