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Tunicamycin

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Tunicamycin is a biochemical compound used in research laboratories. It inhibits the biosynthesis of N-linked glycoproteins, which are essential for various cellular processes. Tunicamycin is commonly utilized in cell biology studies to investigate the role of glycosylation in cellular function.

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Lab products found in correlation

Cb 5083, by Cayman Chemical (2 mentions) Bortezomib, by Selleck Chemicals (2 mentions) Mammalian cell extraction kit, by Abcam (1 mentions) Emetine, by Cayman Chemical (1 mentions) Cb 5083, by MedChemExpress (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Thapsigargin, by Abcam (1 mentions) Nms873, by Cayman Chemical (1 mentions) Mln7243, by MedChemExpress (1 mentions) Mouse anti gapdh, by Merck Group (1 mentions) Mouse anti v5, by Thermo Fisher Scientific (1 mentions) Rabbit anti trka, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions) Kira6, by Cayman Chemical (1 mentions) Stf 083010, by Axon Medchem (1 mentions) Bafilomycin a1, by AppliChem (1 mentions) Ferrostatin 1 ferr 1, by Merck Group (1 mentions) Sulfasalazine sas, by Merck Group (1 mentions) Desferoxamine dfo, by Merck Group (1 mentions)

6 protocols using tunicamycin

1

Investigating Bag-1 Protein Regulation

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5x105 Bag-1 knock out MCF-7 cells per well were seeded into 6-well plates. 24 hour later, cells were transfected with mock vector, wild-type His- or TAP-tagged Bag-1S or mutants using PEI 25K transfection reagent (Polysciences) with a 1:3 DNA:PEI ratio. Inhibitor treatments were performed at 24 hours post-transfection. For tunicamycin treatment, cells were incubated with 10 μg/ml tunicamycin (AppliChem) for 24 hours. For emetine or CB-5083 treatment, transfected cells were incubated with 25 μM emetine (Cayman) or CB-5083 (MedChemExpress) for 4 hours. DMSO was used as negative control. Cells were harvested and lysed using Mammalian Cell Extraction kit (BioVision). Protein concentration was measured by Bradford (Bio-Rad) assay and equal amounts of cell lysates were analyzed by western blotting.
Can N.D., Basturk E., Kizilboga T., Akcay I.M., Dingiloglu B., Tatli O., Acar S., Ozfiliz Kilbas P., Elbeyli E., Muratcioglu S., Jannuzzi A.T., Gursoy A., Keskin O., Doganay H.L., Karademir Yilmaz B, & Dinler Doganay G. (2021). Interactome analysis of Bag-1 isoforms reveals novel interaction partners in endoplasmic reticulum-associated degradation. PLoS ONE, 16(8), e0256640.
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2

Xenopus oocyte expression of BGT-1 mutants

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Stage V and VI oocytes from Xenopus laevis (Nasco, Fort Atkinson, WI) were separated by an overnight treatment with collagenase (Typ CLS II, Biochrom, Berlin, Germany), subsequent washings in calcium-free ORi and maintained at 16 – 18°C in ORi containing again a calcium concentration of 2 mM. One day after removal from the frog, oocytes were injected either with 23 nl 1mg/2ml tunicamycin (AppliChem, Darmstadt, Germany) solved in ORi or 23 nl ORi alone approximately 60 min prior to injection of cRNA coding either for the wildtype BGT-1 or the mutants. An equivalent amount of ORi was injected as a control (mocks). tunicamycin inhibits enzymes involved in the first steps of N-linked glycoprotein synthesis in the endoplasmic reticulum (ER). Oocytes were maintained at 16–18°C in ORi supplemented with 50 μM gentamycin and 2.5 mM sodium pyruvate, daily washings and discarding of damaged oocytes.
Schweikhard E.S., Burckhardt B.C., Joos F., Fenollar-Ferrer C., Forrest L.R., Kempson S.A, & Ziegler C. (2015). Role of N-glycosylation in renal betaine transport. The Biochemical journal, 470(2), 169-179.
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3

Inducing and Inhibiting ER Stress Pathways

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To induce ER stress, cells were exposed to 5 µg/mL tunicamycin (TM, Applichem, Darmstadt, Germany, #A2242), 100 nM thapsigargin (Tg, Abcam, Cambridge, UK, #ab120286), or 10 nM bortezomib (BZ, Selleckchem, Cologne, Germany, #S1013) for the indicated period. 20 µM of the E1 ubiquitin-activating enzyme inhibitor MLN7243 (MedChemExpress, Junction, NJ, USA, #HY-100487) was used to prevent ubiquitination.
For the cycloheximide (CHX) chase experiments, HMC-1.2 cells were treated with 50 μg/mL CHX (Sigma-Aldrich, #01810) for the indicated times. VCP activity was inhibited by CB-5083 (#19311) or NMS-873 (#17674) (1 μM; both from Cayman, Ann Arbor, MI, USA).
Ahmed N., Preisinger C., Wilhelm T, & Huber M. (2024). TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2. Cells, 13(9), 747.
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4

TRKA Phosphorylation and Glycosylation Analysis

Cited 1 time
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Western blotting has been described previously [70 (link)]. Antibodies used included: rabbit anti-TRKA (#06-574; 1:1000) and mouse anti-pY (#05-1050; 1:2000) from Millipore; rabbit anti-TRK (#4609; 1:500) and rabbit anti-phospho-TRKA (#9141; 1:1000) from Cell Signaling; mouse anti-V5 (#R960-25; 1:5000) from Invitrogen; mouse anti-GAPDH (#G8795; 1:5000) from Sigma-Aldrich.
N-linked glycosylation was inhibited from 6 h after transfection by adding tunicamycin (2 µg/ml; AppliChem) and cells were lysed 9 h later in RIPA buffer.
Immunoprecipitation, immunofluorescence and confocal microscopy were done as described previously [70 (link)].
Luberg K., Park R., Aleksejeva E, & Timmusk T. (2015). Novel transcripts reveal a complex structure of the human TRKA gene and imply the presence of multiple protein isoforms. BMC Neuroscience, 16, 78.
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5

Evaluating Novel Pharmacological Inhibitors

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Tunicamycin was purchased from Applichem, Bortezomib from Selleckchem, STF-083010 from Axon Medchem, KIRA6 from Cayman, and GSK2606414 as well as JNK-IN-8 from Calbiochem. DMSO was obtained from Carl Roth GmbH & Co. MKC-8866 was provided by MannKind Corporation, Valencia, CA USA.
Wilhelm T., Bick F., Peters K., Mohta V., Tirosh B., Patterson J.B., Kharabi-Masouleh B, & Huber M. (2017). Infliction of proteotoxic stresses by impairment of the unfolded protein response or proteasomal inhibition as a therapeutic strategy for mast cell leukemia. Oncotarget, 9(3), 2984-3000.
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6

Preparation of Chemical Compounds for Research

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Sulfasalazine (SAS) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Sulfasalazine was dissolved in 400 mM ammonium hydroxide under sterile conditions to concentration of 200 mM. Bafilomycin A1 and Tunicamycin were purchased from AppliChem (Darmstadt, Germany). Bafilomycin A1 was diluted with DMSO to a stock of 200μM and Tunicamycin in DMSO to 2,5 mg/ml. Desferoxamine (DFO) and Ferrostatin-1 (Ferr-1) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Desferoxamine was dissolved in water under sterile conditions to a concentration of 50 mM. Ferr-1 was prepared in 50% DMSO/water under sterile conditions to a concentration of 50 mM.
Sehm T., Fan Z., Ghoochani A., Rauh M., Engelhorn T., Minakaki G., Dörfler A., Klucken J., Buchfelder M., Eyüpoglu I.Y, & Savaskan N. (2016). Sulfasalazine impacts on ferroptotic cell death and alleviates the tumor microenvironment and glioma-induced brain edema. Oncotarget, 7(24), 36021-36033.
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