4.9. Western Blot Analysis in Brain Tissues

Mouse brain (n = 5/group, left hemispheres) was homogenized in RIPA cell lysis buffer (Sigma-Aldrich) with protease inhibitors. Proteins were obtained by centrifugation at 15,000 rpm at 4 °C for 15 min and quantified by the BCA protein assay kit (Pierce, Rockford, IL, USA). Proteins were denatured by heating for 5 min at 95 °C in 0.5 M Tris-HCl buffer (pH 6.8) containing 10% SDS and 10% ammonium persulfate, separated by electrophoresis on SDS-polyacrylamide gel, and transferred onto a polyvinylidene difluoride membrane in 25 mM Tris buffer containing 15% methanol, 1% SDS, and 192 mM glycine. After blocking for 2 h with 5% skim milk in TBS-Tween (TBS-T; 20 mM Tris, 137 mM NaCl, 0.1% Tween 20, pH 7.6), the membrane was incubated with antibodies specific for brain-derived neurotrphic factor (BDNF; 1:500, rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), nerve growth factor (NGF; 1:500, rabbit polyclonal, Santa Cruz Biotechnology), ciliary neurotrophic factor (CNTF; 1:500, rabbit polyclonal, Santa Cruz Biotechnology), neurotrophin 3 (NT3; 1:500, goat polyclonal, Santa Cruz Biotechnology), glial cell-derived neurotrophic factor (GDNF; 1:500, rabbit polyclonal, Santa Cruz Biotechnology), tropomyosin-related kinase B (TrkB; 1:500, rabbit polyclonal, Santa Cruz Biotechnology), p-AKT1/2/3 (1:500, mouse monoclonal, Santa Cruz Biotechnology), calcium/calmodulin-dependent protein kinase II (CaMKII; 1:500, mouse monoclonal, Santa Cruz Biotechnology), protein kinase C (PKC; 1:500, mouse monoclonal, Santa Cruz Biotechnology), extracellular signal-regulated kinase (p-Erk; 1:500, rabbit polyclonal, Santa Cruz Biotechnology), β-site amyloid precursor protein-cleaving enzyme (BACE; 1:500, rabbit polyclonal, Santa Cruz Biotechnology) or neprilysin (1:500, rabbit polyclonal, Santa Cruz Biotechnology) overnight at 4 °C. After washing with TBS-T, the membrane was incubated with a secondary goat anti-rabbit IgG (1:2000, Santa Cruz Biotechnology), rabbit anti-goat IgG (1:2000, Santa Cruz Biotechnology) or goat anti-mouse immunoglobulin G (IgG; 1:2000, Santa Cruz Biotechnology) conjugated with horseradish peroxidase for 2 h at room temperature [9,10]. Then, the membrane was developed using an ECL solution (Pierce).