Colorimetric Assay for CN Activity

CN activity was measured following a modification of the procedures as previous studies.^{49 (link)} Briefly, the basal wells contained 25 mM MOPS (pH 7.0), 2 mM p-nitrophenol phosphate (pNPP), 1 mM DTT, 2 mM EGTA, and 2 mM EDTA. Other than that, the maximum wells contained 2 mM MnCl₂. The final volumes of all wells were 100 μL. Brain region homogenate (200 μg/mL) was added to start the reaction at 37 °C for 0.5 h. The reaction was then terminated by placing on ice and OD was measured at 405 nm. The absorbance units were converted to the concentrations of p-nitrophenol (pNP) through comparing with a standard absorption curve of pNP concentration.