Western Blotting of Brain Tissue Proteins

Western blotting was performed as described previously^{36 (link)}. Briefly, hippocampus and cerebral cortex tissues were homogenized in an ice-chilled RIPA Buffer (FUJIFILM Wako Pure Chemical Corporation, Chuo, Osaka, Japan) containing protease inhibitors (cOmplete, Sigma-Aldrich Japan K.K., Meguro, Tokyo, Japan). The lysate was electrophoresed on a 10% Bis–Tris gel in 1× MOPS buffer. After transfer onto polyvinylidene fluoride membranes, nonspecific antibody binding was blocked for 1 h at room temperature using ImmunoBlock (DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan). Membranes were incubated for 24 h at 4 °C with primary antibody in Can Get Signal Immunoreaction Enhancer Solution (Toyobo Co., LTD., Kita, Osaka, Japan). Anti-PRKAR1B antibody (PA5-87652, Thermo Fisher Scientific K.K., Minato, Tokyo, Japan) was used at a dilution of 1:2000, and anti-actin antibody (A3853; Sigma-Aldrich, St. Louis, MO, USA) was used at 1:10,000. The secondary antibody was an anti-rabbit IgG horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) and was used at 1:100,000 in Can Get Signal Immunoreaction Enhancer Solution. Signals were developed by ECL Western Blotting Detection Reagents (GE Healthcare) and detected using an ImageQuant LAS 4000 Image Analyzer (GE Healthcare Japan Corporation, Hino, Tokyo, Japan).