Protein Expression Analysis in Hippocampal Tissue

Western blot analysis for NF-κB, IκB-α, Bax, Bcl-2, MMP-9 expression was used as explained below (Ko et al., 2020 (link); Park et al., 2020 (link)). Hippocampal tissues were lysed in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH, 7.5), 1 mM phenylmethylsulfonyl fluoride, 100-mg/mL leupeptin, 1% Nonidet P40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate. Rabbit NF-κB antibody (1:1,000; Abcam), rabbit IκB-α antibody, rabbit phosphorylated IκB-α (p-IκB-α) antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit MMP antibody (1:2,000; Cell Signaling Technology, Inc., Danvers, USA), mouse Bax antibody, mouse Bcl-2 antibody, and β-actin antibody (1:1,000; Santa Cruz Biotechnology) were used as the primary antibodies. Horseradish peroxidase-conjugated anti-mouse antibody (1:2,000; Vector Laboratories, Burlingame, CA, USA) for β-actin, Bax, Bcl-2, and horseradish peroxidase-conjugated anti-rabbit antibody (1: 2,000; Vector Laboratories) for NF-κB, IκB-α, and MMP were used as the secondary antibodies. Image-Pro Plus computer-aided image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA) was used for band quantification.