Histone acetyltransferase activity assay

HAT activity assay was performed using the HAT Activity Fluorometric Assay Kit (BioVision, Milpitas, CA, USA). Nuclear extracts were isolated with the Nuclear/Cytosol Fractionation Kit (BioVision) as per the manufacturer’s instructions. For sample preparation, 10 μl of sample was obtained and mixed with HAT Assay Buffer to reach a volume of 50 μl. One well filled with 50 μl HAT Assay Buffer was taken as background control. Then 4 μl of HeLa Nuclear Extract was mixed with HAT Assay Buffer to reach a volume of 50 μl. The mixture was added into desired wells for positive control. Standard Curve Preparation and Reaction Mix were made as per the manufacturer’s instructions. Then, 50 μl of the reaction mix was added to each well containing the samples, background control, standards and positive control. Fluorescence at Ex/Em = 535/587 nm were measured in kinetic mode with a microplate reader (BioTek, Winooski, VT, USA) at 25°C for 60 min for the samples and background control, and the corresponding relative fluorescence unit (RFU) were obtained.