Hdac activity assay kit

Sodium butyrate, propionate, trichostatin A (TSA, a selective and reversible hydroxamate inhibitor of class I and II HDACs) (Hebbel et al., 2010 (link)), sodium β-hydroxybutyrate (SHB, antagonist for GPR41 receptor) (Kimura et al., 2011 (link)), and LPS (Escherichia coli 0111:B4) were purchased from Sigma-Aldrich, St. Louis, MO, United States. Sodium acetate was bought from Merck Millipore. A cytotoxicity detection kit (lactate dehydrogenase, LDH) was obtained from Roche. Human IL-6, IL-8 ELISA (enzyme-linked immunosorbent assay) kits, and calcein-AM were purchased from Invitrogen. Human recombinant TNFα, anti-human CD106 (VCAM-1) PE, and viability fixable dyes were bought from eBioscience. GLPG0974 (GLPG, antagonist of GPR43 receptor) (Namour et al., 2016 (link)) was obtained from Tocris Bioscience. Primary anti-GPR41 antibody, anti-GPR43 antibody, and an HDAC activity assay kit were purchased from Abcam. EGM-2 Bulletkit was purchased from Lonza (Switzerland).

HDAC activity was measured with the fluorometric HDAC Activity Assay kit (Abcam, Cambridge, MA) according to the manufacturer’s instruction. Briefly, the cell lysates with or without TSA treatment were sonicated, cleared, and incubated with assay buffer containing the HDAC substrate [Boc-Lys(Ac)-AMC] for 30 min at 37 °C. The reaction was terminated, and the fluorescence intensity was measured in a fluorescence plate reader (Ex/Em = 350–380/440–460 nm).

HDAC activity was measured in total cell lysates or after immunoprecipitation with isoform-specific antibodies using the fluorometric HDAC Activity Assay kit (Abcam, Cambridge, MA) as previously described [35 (link)]. Briefly, cell lysates or immunoprecipitates were incubated with assay buffer containing 0.2 mM Boc-Lys(Ac)-AMC as a substrate for 30–60 min. Subsequently, trypsin was added with 1 μM trichostatin A (TSA) to terminate the deacetylation and cleave deacetylated substrates. Fluorescence was measured with excitation at 390 nm and emission at 460 nm.
For the tubulin deacetylation assay, cells transiently expressing Flag-tagged HDAC6 were lysed in a buffer of 50 mM Tris–HCl, pH 7.6, 120 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 with protease inhibitors and tagged HDAC6 was immunoprecipitated with anti-Flag (M2) antibody. After several washes in assay buffer (10 mM Tris–HCl, pH 8.0, 10 mM NaCl), immunoprecipitated proteins were incubated with 10 μg of microtubule associated protein (MAP)-stabilized microtubules [polymerized from a MAP-rich tubulin fraction (Cytoskeleton, Inc)] for two hours at 37°C. Supernatant and beads were separated by centrifugation and subjected to immunoblotting as described [36 (link)].

The MzChA-1 cells were cultured with or without 5 ng/ml TGF-β1 and 300 nM vorinostat for 72 h. Then, nuclear proteins were extracted with the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Subsequently, the activity of HDAC was measured with the HDAC activity assay kit (BioVision, Palo Alto, USA). In this experiment, we used HeLa nuclear extract as a positive control and distilled water as a negative control. All procedures were conducted according to the manufacturer’s recommendations.