Membrane preparation

Rats were sacrificed, and the cerebral cortex, hippocampus, and striatum were dissected quickly. Rat or human brain tissue was homogenized in 5 mL of ice-cold TED buffer (5 mM Tris–HCl, 1 mM EDTA, 1 mM dithiothreitol; pH 7.4) containing 10% (w/v) sucrose by 20 strokes with a motor-driven Teflon/glass tissue grinder. All of the following centrifuge procedures were performed at 4 °C. Subsequent to centrifugation of the homogenate at 1000 g for 10 min, the supernatant was decanted to another centrifuge tube. The pellet was vortexed in 5 mL of TED/sucrose buffer and centrifuged again at 1000g for 10 min. The combined supernatant (10 mL) was centrifuged at 9000g for 20 min and resuspended in 10 mL of TED buffer. After the same procedure was repeated, the homogenate was kept on ice for 30 min, followed by a final centrifugation at 35,000g for 10 min. The resulting pellet was resuspended in 50 mM Tris–HCl buffer (pH 7.4) to produce the homogenate with a protein concentration of 1.0–2.0 and 2.0–3.0 mg/mL for rat and human brain, respectively. The homogenate was frozen quickly on fine-grained dry ice and stored at − 80 °C until the day of experiment.