Total protein was conducted from the frozen tissues of cerebral cortex using RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1% protease inhibitors. Nucleoprotein extraction was conducted using nuclear and cytoplasmic protein extraction kit (product code: P0028; Beyotime Biotechnology, Inc, Shanghai, China). Then, the protein concentration was determined by BCA Protein Assay Kit (Beyotime, P0010). And an equal amount of protein from each sample was separated by SDS-PAGE gels and transferred onto the PVDF membranes. After blocking with 5% milk in TBST for 2 h, the membranes were reacted overnight at 4℃ with antibodies, including anti-β-Actin antibody, anti-Histone H3 antibody, anti-NF-κB p65 antibody, anti-TNF-α antibody and anti-Cox-2 antibody. Subsequently, the membranes were washed extensively and incubated with Goat anti-rabbit IgG (Bioworld Technology, USA, 1:1000). Finally, the level of protein was determined and analysed by Image Lab (Bio-Rad Laboratories, USA). We used Histone H3 as an internal control for nucleoprotein loading and β-Actin as an internal control for total protein loading to normalize each sample.

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