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Cells were lysed in a protein lysate buffer (50 mM Tris pH 7.4, 100 μM EDTA, 0.25 M sucrose, 1% SDS, 1% NP40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A and 100 μM phenyl methyl sulfonyl flouride) and homogenized. Debris was removed by centrifugation at 10,000 × g for 10 min at 4 °C, and protein concentrations were measured ATPase Activity Assay Kit (Sigma-Aldrich; malachite green assay, MAK113) following the manual provided by the manufacturer. Absorbance was read at 620 nm, and free phosphate was calculated.
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