Hat activity colorimetric assay kit

The analysis of the methylation or acetylation status of histones H3 and H4 N-terminal tails was performed by Western blotting, following the protocol described above. The post-translational modifications were assessed by using specific antibodies to detect histone methylation (H3K27me3 and H3K9me3) or histone acetylation (H3Ac e H4Ac). In addition, the activity of thyroid HAT and HDAC enzymes were evaluated by using a HAT Activity Colorimetric Assay Kit and a HDAC Activity Assay Kit, respectively, following protocol indicated by the manufacturer (Sigma Aldrich, St. Louis, MO, USA).

In vitro α-tubulin acetylation assay was performed as previously described^{30 (link)}. Briefly, 96-well half-area plates (Greiner, 674061) were coated with 5 µg tubulin from HeLa cells or bovine brain (Cytoskeleton, catalog no. HTS02-A) in 25 µL of ultrapure water for 2.5 h at 37 °C, blocked (PBS + 3% BSA + 3% skim milk + 3% FBS) for 1 h at 37 °C, and washed with PBS + 0.05% Tween 20. Wells were incubated for 2 h at 37 °C with shaking at 140x g with 25 µg of the total, S2, S3, or P3 fractions isolated from P0 mouse brain cortex previously diluted in 2× histone acetyltransferase (HAT) buffer (Sigma–Aldrich, EPI001A) supplemented with protease inhibitor cocktail, 5 µM trichostatin, and 50 µM acetyl-CoA (Sigma-Aldrich, A2056) or vehicle (H20) for control. After washes and overnight incubation at 4 °C with acetylated a-tubulin antibody (1:2000) in blocking buffer (PBS + 0.05% Tween 20 + 3% BSA), wells were washed and incubated for 2 h at 37 °C with peroxidase-conjugated goat antimouse antibody (1:5000) in antibody blocking buffer. Wells were then washed and incubated with trimethylboron/E (Merck Millipore, ES001) whose reaction was thereafter stopped with H2SO4. Following the manufacturer’s instructions, the HAT activity colorimetric assay kit (Sigma–Aldrich, EPI001) was used to measure CoA release.