2.7. Na, K-ATPase activity assay

Enzyme activity was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP conversion using a high throughout colorimetric ATPase assay kit from Innova Biosciences (601–0120) and follows the protocol of Fishburn et al. (2015). In brief, 40-μL reactions contained 10 mM Hepes (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 3.5% glycerol, 1 mM DTT, 4 μg BSA, 10 ml lysate (cell or tissue standardized as 10 μg/ml). After 40 min at room temperature, purified ATP was added to 0.5 mM, and reactions were incubated 1–20 min at 26 °C. Reactions were stopped by the addition of 10 μl gold mix and, after 4 min, 4 μl stabilizer 2. After 30 min at room temperature, absorbance was measured at 600 nm and plotted against protein concentration. A standard curve was established for every experiment using the phosphate standard.