NKA activity assay

Brain and cell samples were lysed in buffer A (20 mM Hepes, 250 mM sucrose, 2 mM EDTA, and 1 mM MgCl₂, pH 7.4). The pellet was resuspended in buffer A, and the protein concentration was determined with a BCA assay kit (Pierce, USA). Samples were divided into two aliquots: One aliquot (50 μl) was incubated with reaction buffer 1 [50 μl, 200 mM tris (pH 7.5), 30 mM MgCl₂, 200 mM NaCl, 60 mM KCl, and 10 mM EGTA], and the other aliquot (50 μl) was incubated with reaction buffer 2 (buffer 1 + 1 mM OB; Sigma-Aldrich, O3125). ATP (1 mM) was added to start the reactions at 37°C for 10 min. After terminating the reactions by adding trichloroacetic acid [10 μl, 100% (w/v)], samples were placed on ice for 1 hour. Free phosphates were then collected in the supernatant of the samples after centrifuging at 20,000g for 30 min. Phosphate colorimetric kit (Sigma-Aldrich, MAK030) was used to measure the free phosphates. The enzyme activity of NKA was defined as the difference of absorbance at 650 nm between the two aliquots.