Tubastatin a

Manufactured by Merck Group

Sourced in United States

Tubastatin A is a synthetic small molecule that functions as a selective inhibitor of histone deacetylase 6 (HDAC6). It is used as a research tool to study the biological role of HDAC6 in various cellular processes.

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Tubastatin (4 citations)

19 protocols using tubastatin a

Isolation and Osteogenic Differentiation of Mesenchymal Stem Cells

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The bone marrow from femur and tibia were suspended in cold PBS with 2% FBS and passed through a 70 μm filter. Filtered bone marrow cells were suspended in PBS with 2% FBS and 0.1 g/L phenol red and then enriched for lineage negative (Lin−) cells using the SpinSep system (Stem Cell Technologies, Vancouver, BC, Canada). The cells were incubated with a murine progenitor enrichment cocktail (Stem Cell Technologies) on ice for 30 min, washed, and then incubated with dense particles on ice for 20 min. The cells were then centrifuged at 1200g for 10 min, and the cells at the density medium/PBS interface were collected.
Enriched MSCs were seeded onto culture plates at a density of 0.1 × 10⁶ cells/cm² in α-MEM containing 100 units/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco). The media were changed after 72 h and adherent cells were maintained in culture with twice weekly media changes.
To induce osteogenic differentiation, BMSCs were treated with 100 nM dexamethasone, 10 mM β-glycerophosphate disodium and 50 μg/ml ascorbic acid.
BMSCs were treated in vitro with tubastatin A (Sigma) in DMSO at 8 μM for 21 days. Vehicle controls were treated with culture medium with DMSO only.

Ma C., Gao J., Liang J., Dai W., Wang Z., Xia M., Chen T., Huang S., Na J., Xu L., Feng S., Dai K, & Liu G. (2021). HDAC6 inactivates Runx2 promoter to block osteogenesis of bone marrow stromal cells in age-related bone loss of mice. Stem Cell Research & Therapy, 12, 484.

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Molecular Mechanisms of Oxidative Stress Regulation

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N-acetyl cysteine (NAC), diphenylene iodonium (DPI), apocynin (APO), and tubastatin A (TBA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dihydroethidium (DHE) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Primary antibodies against gp91phox/NOX2, p47phox, acetylated α-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and those against HDAC6 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 3000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). A luciferase assay kit was purchased from Promega (Madison, WI, USA). Oligonucleotide primers (HDAC6, CCL2, CXCL8, CXCL10, Nox2, p22phox, p47phox, and β-actin) and HDAC6 siRNA were obtained from Bioneer (Seoul, Korea).

Youn G.S., Cho H., Kim D., Choi S.Y, & Park J. (2017). Crosstalk between HDAC6 and Nox2-based NADPH oxidase mediates HIV-1 Tat-induced pro-inflammatory responses in astrocytes. Redox Biology, 12, 978-986.

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Tubastatin A Effects on Mouse Bone Homeostasis

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Male C57BL/6 mice (SIPPR-BK Laboratory Animal Co. Ltd, Shanghai, China) were housed under SPF conditions. All animal operations were approved by the Animal Ethics Committee of Xuzhou Central Hospital (Animal Protocol No. XZZX-LJ-20190201-18)
12 animals were included per group. Animals were euthanized using isoflurane inhalation anesthesia followed by cervical dislocation. The right femur, tibia and lumbar vertebrae (L5) were prepared for micro computed tomography (micro-CT) scan; the left femur and tibia were used for BMSCs isolation. Peripheral serum was taken for the measurement of osteocalcin and N-telopeptide (Biomedical Technologies, Stoughton, MA).
Animals were administrated intraperitoneally with tubastatin A (Sigma Chemical Co., St. Louis, MO) in 0.9% saline with 4% DMSO and 30% PEG300 at dose of 8 mg/kg once every two days for 30 days. After 30 days, animals were euthanatized for micro-CT and serum biomarkers analysis.

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Assessing Effects of Pharmacological Agents on Edema in stk3+/- Zebrafish

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Embryos were obtained with stk3^+/− in-cross and distributed randomly to different treatment groups. Drugs or vehicle control were added to fish water at 8 dpf and changed every other day. All treatments were initiated at 8 dpf and ended at 20 dpf. The larvae were monitored twice a day, and individuals with edema were recorded and genotyped. The remaining individuals were all genotyped at the end of the experiment.
Verteporfin, dexamethasone, prednisolone, rapamycin and tubastatin A were all purchased from Sigma-Aldrich and dissolved in DMSO as stocks. All treatment and vehicle control groups contained 0.001% DMSO.

Ren Z., Zhang Z., Liu T.M, & Ge W. (2021). Novel zebrafish polycystic kidney disease models reveal functions of the Hippo pathway in renal cystogenesis. Disease Models & Mechanisms, 14(11), dmm049027.

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Generation and Characterization of Fluorescent Fusion Protein Constructs

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Full-length cDNAs for RAPGEF2 and Bcl-2-associated X protein (BAX) were PCR-amplified from total human cDNAs and cloned into the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) to generate pEGFP-RAPGEF2 and pEGFP-BAX. The E1357K mutation was introduced into pEGFP-RAPGEF2 by PCR-mediated mutagenesis to produce pEGFP-RAPGEF2-E1357K.
Primary human fibroblasts were established from punch biopsies on the forearm skin of the patient and a 43-year-old male control as described previously [23 (link)] and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 20% heat-inactivated (30 min, 55℃) fetal bovine serum (FBS), 1% non-essential amino acids, and antibiotics. Passage-matched control and patient fibroblasts (prior to passage 10) were used in each experiment. For inhibition of HDAC6, human skin fibroblasts were treated with 1 µM tubastatin A (Sigma-Aldrich, St. Louis, MI, USA) overnight at 37℃. Human HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and transfected using FuGENE HD transfection reagent (Promega, Madison, WI, USA).

Heo K., Lim S.M., Nahm M., Kim Y.E., Oh K.W., Park H.T., Ki C.S., Kim S.H, & Lee S. (2018). A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution. Experimental Neurobiology, 27(6), 550-563.

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Cardiac Electrophysiology Assay Using hiPSC-CMs

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All drugs were obtained from Sigma Aldrich: vanoxerine (Cat. D052), cisapride (C4740), dofetilide (PZ0016), nifedipine (N7634), as well as the following histone deacetylase inhibitors, HDACi: 4-phenylbutyrate, 4PB (567616), tubastatin A (SML0044) and from a kit (EPI009-1KT): trichostatin-A (TSA), panobinostat, and SAHA. The drugs were dissolved in DMSO (Cat. 34869, Sigma-Aldrich), except for 4PB (dissolved in sterile water) and stored in −20°C until ready for application on the hiPSC-CMs. The HDAC inhibitors TSA, panobinostat, 4PB, tubastatin, and SAHA stock solutions were diluted in hiPSC-CM maintenance media and applied to the cells 48 hours prior to functional measurements. vanoxerine, cisapride, dofetilide, and nifedipine stocks were diluted in Tyrode’s solution on experimental day and applied for 30 minutes at 37°C, 5% CO2 prior to functional experiments. Drug concentrations are as specified in the figures, and all were selected to be at 1X to 10X of the effective free therapeutic plasma concentrations, ETPC, see Table 1.

Heinson Y.W., Han J.L, & Entcheva E. (2023). OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology. bioRxiv.

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Hemorrhagic Shock Treatment Evaluation

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After shock, all animals were randomly allocated to the following groups (n = 8 per group): (1) sham (anesthetized and cannulated but without hemorrhage or any treatment), (2) no treatment (hemorrhage but without any treatment, negative control), (3) vehicle (dimethyl sulfoxide [DMSO]: 1 mL/g; Sigma-Aldrich), (4) MS-275, (5) LMK-235, (6) tubastatin A, (7) TSA, and (8) sirtinol. All reagents were obtained from Selleckchem, Houston, TX, and were dissolved in DMSO. Drugs were administered at the end of the shock (MAP was maintained at 30–40 mm Hg) via i.v. over 20 min.

Niu K., Yang L., Song W., Liu Z., Yuan J., Zhang H., Zhang W., Wang J, & Tao K. (2023). A COMPARATIVE ANALYSIS TO DETERMINE THE OPTIMUM HISTONE DEACETYLASE INHIBITORS AND ADMINISTRATION ROUTE FOR IMPROVING SURVIVAL AND ORGAN INJURY IN RATS AFTER HEMORRHAGIC SHOCK. Shock (Augusta, Ga.), 60(1), 75-83.

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CD34+ Cell Expansion with HDAC6 Inhibitors

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CD34⁺ cells were obtained, in agreement with our institutional ethics committee (Assistance Publique des Hôpitaux de Paris) and in accordance with the Declaration of Helsinki, from leukapheresis samples. CD34⁺ cells were isolated by a positive selection using immunomagnetic bead cell-sorting system (AutoMacs; Miltenyi Biotec) and cultured in serum-free medium in the presence of recombinant human thrombopoietin (rhTPO; 10 ng/mL; Kirin Brewery). Two HDAC6-selective inhibitors, Tubastatin A (Sigma) and ACY1215, generously provided by Simon Jones (Acetylon Pharmaceuticals Inc.), were used.

Messaoudi K., Ali A., Ishaq R., Palazzo A., Sliwa D., Bluteau O., Souquère S., Muller D., Diop K.M., Rameau P., Lapierre V., Marolleau J.P., Matthias P., Godin I., Pierron G., Thomas S.G., Watson S.P., Droin N., Vainchenker W., Plo I., Raslova H, & Debili N. (2017). Critical role of the HDAC6–cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect. Nature Communications, 8, 1786.

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HDAC Deacetylase Activity Assay

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HDAC activity was measured with a homogenous fluorescence release HDAC deacetylase assay. EVs (supernatant from 1 compartment per transwell plate) or cell lysates (3 μg protein) were incubated with (AMC)-K(Ac)GL-Ac substrate (synthesized as published by us, >95% purity, [31 (link)]) to assess class I HDACs (HDACs 1, 2, 3, 6 and 10) enzyme activity in the presence of MS-275 (inhibitor of HDAC 1, 2 and 3 activities [32 (link)], Selleckchem). Deacetylated AMC-KGL is sensitive toward lysine peptidase (1 mg/ml trypsin), generating free fluorogenic 4-methylcoumarin-7-amide (excitation 355 nm, emission 460 nm; Fluoroskan Ascent Microplate Fluorometer [Labsystems]). Assay conditions were set with excess substrate concentrations to ensure linear deacetylation kinetics. Data were standardized using the control, and absolute deacetylated substrates were calculated based on the standard curve generated by nonacetylated AMC-KGL substrate under the same conditions [33 (link)].
HDAC6 activity analysis in recipient monolayers was confirmed using inhibitors Trichostatin A (TSA; Sigma) and Tubastatin A (Tub A; Sigma) at concentrations indicated in the text.

Shah N., Ishii M., Brandon C., Ablonczy Z., Cai J., Liu Y., Chou J, & Rohrer B. (2018). Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers.. Biochimica et biophysica acta. Molecular basis of disease, 1864(8), 2610-2622.

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Oxidative Stress Biomarkers in Neurodegenerative Models

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6-OHDA hydrobromide, tubastatin A, and sodium hydrogen sulfide were obtained from Sigma Aldrich. RNA isolation kits, cDNA synthesis, and SYBR Green/ROX master mix were from Qiagen. Primer sequences for PCR were obtained from Eurofins MWG (Operon). Protein carbonyl, lipid peroxidation, superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) assays kits were obtained from Cayman Chemicals, USA. The HDAC activity assay kit was obtained from Biomol, PA, USA. The HAT activity assay kit was acquired from Active Motif, Carlsbad, CA, USA. The Fluoro-Jade C assay kit was obtained from Millipore Sigma, Burlington, MA, USA. GFAP monoclonal antibody and the corresponding anti-mouse secondary antibody were obtained from Santa Cruz Biotechnology, USA. ELISA kits for IL-1β (ER1094), IL-2 (ER0039), IL-17 (ER0035), IL-8 (ER1623), IL-6 (ER0042), TH (ER0534), GFAP (ER0229), α-synuclein (ER0921), and TNF-α (ER1393) were obtained from Fine Biotech, China. All other chemical reagents used were analytical grade.

Sun Y., Li D., Su Y., Zhao H., Pang W., Zhao W, & Wu S. (2020). Protective effect of hydrogen sulfide is mediated by negative regulation of epigenetic histone acetylation in Parkinson’s disease. Archives of Medical Science : AMS, 19(4), 1124-1135.

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