To measure the activity of both HDAC and HAT, the HDAC Activity Assay Kit (Millipore, item #17–356) and HAT Activity Assay Kit (BioVision, item #K334-100) were used according to the manufacturer's instructions. Proteins were extracted from fresh brain tissue and quantified. HDAC and HAT activity was estimated using 20 μg of protein. Briefly, for the HDAC activity assay, the HDAC assay buffer containing TSAs was used as an inhibitor control, HeLa nuclear extract was used as a positive control, and the HDAC assay buffer solution was used as the negative control. Subsequently, the enzyme plate was placed in an ELISA reader for the assay, with excitation light at 350–380 nm and emission light at 460 nm. The detected optical density value's magnitude reflected the HDAC activity level.
For the determination of the HAT activation, dissolve 20 μg brain tissue proteins in 40 μl of H2O (ultimate amount) and then with 5 μl of specific color substrates 1 and 2 based on a manufacturer's directions. 50 μl of 2 × HAT assay buffer and 8 μl of NADH-generating enzyme were added. After thorough mixing, the samples were incubated at 37 °C for 4 h. Subsequently, the samples were placed in an enzyme calibrator for detection at a reading wavelength of 440 nm. The magnitude of the value obtained reflected the level of HAT activity.
For the determination of the HAT activation, dissolve 20 μg brain tissue proteins in 40 μl of H2O (ultimate amount) and then with 5 μl of specific color substrates 1 and 2 based on a manufacturer's directions. 50 μl of 2 × HAT assay buffer and 8 μl of NADH-generating enzyme were added. After thorough mixing, the samples were incubated at 37 °C for 4 h. Subsequently, the samples were placed in an enzyme calibrator for detection at a reading wavelength of 440 nm. The magnitude of the value obtained reflected the level of HAT activity.