In vitro Kinase assays
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For in vitro HDAC assays, myc epitope-tagged HDAC1 was transfected into HeLa cells and immunoprecipitated. Immunoprecipitates were washed, resuspended in HD buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol), and stored as frozen aliquots. HDAC1 was then added to a tube containing 40,000 cpm 3H-labeled acetylated histones (purified from HeLa cells) in 200 μl of HD buffer ±valproic acid,. After rotation for 2 h at 37 °C, the reaction was stopped by the addition of 50 μl of stop solution and released 3H-labeled acetic acid was extracted and analyzed by scintillation counting.
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References:
1. Phiel, C. J., Zhang, F., Huang, E. Y., Guenther, M. G., Lazar, M. A. and Klein, P. S. (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 276, 36734-36741
2. Shan, Z., Feng-Nian, R., Jie, G. and Ting, Z. (2012) Effects of valproic acid on proliferation, apoptosis, angiogenesis and metastasis of ovarian cancer in vitro and in vivo. Asian Pac J Cancer Prev. 13, 3977-3982
3. Song, R., Yu, D., Yoon, J. and Park, J. (2015) Valproic acid attenuates the expression of pro-inflammatory cytokines lipopolysaccharide-treated canine peripheral blood mononuclear cells (in vitro) and in a canine endotoxemia model (in vivo). Vet Immunol Immunopathol. 166, 132-137
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