Na/K-ATPase Activity Assay

The neuronal membrane and synaptosomes were prepared from mice cortical tissues (4). The collected membrane fractions and synaptosomes were resuspended in buffer with 10 mM Tris-HCl, 150 mM NaCl, pH7.4 for Na/K-ATPase activity assay kit (Nanjing Jiancheng, China). The kit was used to measure inorganic phosphate released from ATP hydrolysis in the medium: 130 mM NaCl, 20 mM KCl, 3 mM MgCl2, 3 mM ATP, and 30 mM HEPES (pH 7.4 at 37°C for 10 min) by calculating Na/K-ATPase activity as the difference between total and ouabain independent ATPase. Ouabain-sensitive Na/K-ATPase activity was calculated as the difference between ATPase activity in the presence and in the absence of 1 mM ouabain (52).