Hat activity colorimetric assay kit

HAT activity was measured using the commercially available colorimetric HAT activity assay kit (Abcam). Briefly, 50 µg of HeLa cell nuclear extract (Santa Cruz Biotechnology) was incubated, with or without carnosol, in HAT assay buffer according to the manufacturer’s instructions. Absorbance was measured at 450 nm. Experiments were carried out in triplicate and repeated three times. Data are represented as mean values ± SEM.

The HDAC and HAT activities in the right upper lung tissues were measured using colorimetric activity assay kits (Colorimetric HDAC Activity Assay Kit [Catalog #K331-100); HAT Activity Colorimetric Assay Kit [Catalog #K332-100); BioVision Inc.,USA], in accordance with the manufacturer’s protocols. Briefly, an HDAC colorimetric substrate was incubated with 80 μg of total nuclear extracts from lung tissues for 60 min at 37°C. Following the incubation, lysine-developer solution was added to generate a measurable chromophore that was analyzed by the absorbance at 405 nm using an ELISA microplate reader (Bio-Rad Laboratories, USA). HeLa cells nuclear extracts (4 μg) were used as a positive control. An HDAC inhibitor, trichostatin A, was used to demonstrate the specificity of the deacetylation activities. For the HAT activity assay, an active nuclear extract as a positive control and acetyl-CoA as a cofactor were utilized. Total nuclear extracts (50 μg) were incubated with acetyl-CoA at 37°C for 120 min. Acetylation of the peptide substrate by active HAT released the free form of CoA, which then served as an essential coenzyme for producing NADH. The produced NADH was detected spectrophotometrically upon reacting with a soluble tetrazolium dye. The activity was analyzed by the absorbance at 440 nm using the above-described ELISA plate reader.