Hdac9

Manufactured by BPS Biosciences

Sourced in United States

HDAC9 is a recombinant protein produced by BPS Biosciences. It is a member of the histone deacetylase (HDAC) family of enzymes, which play a role in the regulation of gene expression.

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3 protocols using hdac9

Quantification of HDAC Activities

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Murine or human CD8⁺ T cells were harvested in the lysis buffer, and 100 µl of cell lysates were subjected to HDAC inhibition by adding 5 mM of SCFAs (or 500 nM TSA) for 15 min. Following initial inhibition of HDACs by SCFAs, the peptide substrate Ac-Arg-Gly-Lys(Ac)-AMC (300 µM, Bachem, Bubendorf, Switzerland) was added to the reaction tubes for 30 min. The cleavage of the deacetylated substrate was achieved by addition of 100 µl of the developer solution (10 mg/ml trypsin in 50 mM Tris-HCl, pH = 8, 100 mM NaCl, 2 µM TSA) for 30 min at 37 °C. The fluorescence intensity of free AMC was measured at Ex/Em = 355 nm/460 nm. For the impact of bacterial supernatants and SCFAs on specific HDAC isoforms, the fluorogenic assay for each HDAC enzyme (HDAC1-3, HDAC4-6, HDAC9) was used (BPS Bioscience). Assays were performed according to the manufacturer’s instructions and all measurements were conducted in triplicate using the Omega series Software V5.5, MARS 3.32 R5.

Luu M., Riester Z., Baldrich A., Reichardt N., Yuille S., Busetti A., Klein M., Wempe A., Leister H., Raifer H., Picard F., Muhammad K., Ohl K., Romero R., Fischer F., Bauer C.A., Huber M., Gress T.M., Lauth M., Danhof S., Bopp T., Nerreter T., Mulder I.E., Steinhoff U., Hudecek M, & Visekruna A. (2021). Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer. Nature Communications, 12, 4077.

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High-Throughput Screening of Epigenetic Modulators

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Four cell lines (human colon cancer cell line HCT116, human embryonic kidney cell line HEK293, human liver cancer cell line HepG2, and human B lymphoma cell line SU-DHL-6), fetal bovine serum (FBS), Eagle's Minimum Essential Medium (EMEM), and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from American Type Cell Collection (ATCC, Manassas, VA, USA). H9-derived human neural stem cells, defined FBS, trypsin-EDTA, fibronectin, basal fibroblast growth factor (bFGF), epidermal growth factor (EGF), KnockOut™ Dulbecco's Modified Eagle Medium (DMEM)/F12, and penicillin-streptomycin, StemPro^® Neural Supplement were acquired from Life Technologies, Carlsbad, CA, USA. Chemicals and epigenetic compound libraries were purchased from Cayman Chemicals (Ann Arbor, MI), Santa Cruz Biotechnology (Dallas, TX, USA), SelleckChem (Houston, TX, USA), and Sigma-Aldrich (St. Louise, MO, USA). HDAC-Glo I/II and CellTiter-Glo reagents were purchased from Promega, Madison, WI. Fluorogenic assay kits for HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10 were acquired from BPS Bioscience (San Diego, CA). White solid bottom 1536-well assay plates were purchased from Greiner Bio-One (Monroe, NC).

Hsu C.W., Shou D., Huang R., Khuc T., Dai S., Zheng W., Klumpp-Thomas C, & Xia M. (2016). Identification of HDAC inhibitors using a cell-based HDAC I/II assay. Journal of biomolecular screening, 21(6), 643-652.

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HDAC Enzyme Activity Assay Protocol

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Enzyme activity measurements of class I and IIa HDACs were performed using a cell‐free system and according to a method previously described.16 We used fluorogenic HDAC1 (#50061), HDAC2 (#50062), HDAC3 (#50063), HDAC4 (#50064), HDAC5 (#50065), HDAC6 (#50076), HDAC7 (#50067), HDAC8 (#50068) and HDAC9 (#50069) enzyme assay kits from BPS Bioscience (San Diego, CA, USA). To test the enzyme activity of LMK235, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μmol/L concentrations were used. For the IC₅₀ calculations, every data point was normalized to the vehicle (100% activity). The normalized data were fitted with a Hill non‐linear curve fit (OrigionPro 9.0). Employing the “Find X from Y” function in OrigionPro 9.0 resulted in the IC₅₀ values (50% activity).

Choi S.Y., Kee H.J., Sun S., Seok Y.M., Ryu Y., Kim G.R., Kee S., Pflieger M., Kurz T., Kassack M.U, & Jeong M.H. (2019). Histone deacetylase inhibitor LMK235 attenuates vascular constriction and aortic remodelling in hypertension. Journal of Cellular and Molecular Medicine, 23(4), 2801-2812.

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