Western Blot Analysis of Brain Proteins

After the mice were euthanized, the prefrontal cortex (PFC) and hippocampus (HIP) of each mouse were separated and collected immediately on ice. Then, the total proteins were extracted by homogenization using lysis buffer (RIPA, P0013K, Beyotime, Shanghai, China). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membranes were incubated with the corresponding specific antibodies for NF-κB, COX-2, NLRP3, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), GAPDH, and β-actin overnight at 4°C, followed by incubation with either HRP-labeled Goat Anti-Rabbit IgG (H + L) or HRP-labeled Goat Anti-Mouse IgG (H + L) for 2 h at room temperature. Images of the resulting bands were obtained after ECL (P0018S, Beyotime, Shanghai, China) and developed by the Amersham imager 680 (General Electric Company, Boston, United States). The band intensities were normalized to β-actin or GAPDH. Information on the antibodies utilized in this study is provided in Supplementary Table S3.