HDAC Enzymatic Activity Assays

For Arabidopsis transgenic plants, total proteins were extracted from 1 g of 2-d-old etiolated seedlings using 2 mL cold protein extraction buffer containing the protease inhibitor. Before HDAC enzymatic activity was measured, protein extracts were incubated with 5 μL GFP-trap beads (Chromotek) to immunoprecipitate GFP-tagged HDA15 overnight at 4°C and washed by the wash buffer (50 mm Tris-HCl pH 7.4, 150 mm NaCl, 10% [v/v] glycerol, and 1% [v/v] CA-630). Immunoblotting was carried out to identify the immunoprecipitation efficiency of GFP-trap beads using the GFP antibody (Abcam, ab290).

HDAC enzymatic activity assays were performed using the Fluorometric HDAC Activity Assay Kit (BioVision) following the manufacturer’s instructions. For Arabidopsis transgenic plants, 103 μL double distilled water, 12 μL 10× HDAC assay buffer, and 5 μL substrate [0.02 mm Boc-Lys(Ac)-7-amino-4-methylcoumarin (AMC)] were used. For kinetics assays, different substrate concentrations were used for the HDA15HD monomer, tetramer, and HDA15ZFHD dimer. The 75 nm protein of monomeric HDA15HD and 25 nm protein of tetrameric HDA15HD were reacted with the substrate, and 2.5 nm protein of dimeric HDA15ZFHD was used. The total reaction volume was 120 µL, and the deacetylation reaction was incubated at 37°C for 30 min. The reaction was stopped by adding 80 μl Lys developer and incubated at 37°C for 30 min.

The HDAC activity was then measured using the RFU (Relative fluorescence unit) by ELISA reader (TECAN Infinite M200, Ex/Em = 350/440 nm) on a 96-well black plate. A standard curve was prepared using the AMC Standard (range from 0 to 2 μm). AMC released was used to stand for HDAC enzymatic activity. For kinetics assays, the results were further transformed to molecular concentration for enzyme kinetics calculations.