Decanoyl rvkr cmk

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Decanoyl-RVKR-CMK is a synthetic peptide that functions as a protease inhibitor. It specifically inhibits the activity of furin, a proprotein convertase enzyme involved in the proteolytic cleavage of precursor proteins.

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5 protocols using decanoyl rvkr cmk

Investigating Proprotein Convertase Inhibition in Interleukin-Mediated Cell Priming

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Cells of two human HRS-positive cell lines (T-47D breast cancer cells and T98G glioblastoma cells) were used. The culture conditions were as described previously [4 , 10 (link), 19 (link)] using RPMI1640 (Roswell Park Memorial Institute) medium (JRH Biosciences, Kansas, USA) supplemented with 10% fetal calf serum (Gibco, Paisley, UK), 2 mM L-glutamine (SIGMA, St Louis, MO, USA), 200 units l⁻¹ insulin (SIGMA), and 1% C. The cells were kept in exponential growth by reculturing of stock cultures two times a week. The cells were tested negative for the presence of mycoplasma.
In experiments with interleukins 4 or 13 (IL4–10H and IL13–22H, Creative Biomart, NY 11967, USA), the cells were washed three times with serum-free medium before serum-free medium with 10 ng/ml interleukin was added for cell conditioning and LDR priming. fetal calf serum (10%) was added 1 h after the end of irradiation.
Decanoyl-RVKR-CMK (Tocris Bioscience, Bristol, BS11 0QL, UK) (20 μM) was used to block the activity of all seven proprotein convertases (PC1, PC2, PC4, PACE4, PC5, PC7 and furin). It was added together with 10 ng/ml IL-13 in serum-free medium to the cells 4 h before LDR priming. The medium was exchanged with normal full medium 1 h after the end of irradiation.
IL-13, IL-13Rα2 and TGF-β3 neutralizing antibodies were purchased from R&D (R&D systems, Minneapolis, MN, USA).

, & Edin N.F. (2014). The role of interleukin-13 in the removal of hyper-radiosensitivity by priming irradiation. Journal of Radiation Research, 55(6), 1066-1074.

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Huh-7 Cells Inhibitor Pretreatment

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Huh-7 cells were incubated with 10 μM decanoyl-RVKR-CMK (Tocris), 50 μM Camostat (MedChemExpress), or 50 μM E64D (Selleck) for 2 h before infection. Equal volumes of water and dimethylsulfoxide (DMSO) served as vehicle controls. After the 2-h pretreatment, the media containing inhibitors and control media were removed. The Huh-7 cells were infected with MjHKU4r-CoV-1 at a multiplicity of infection (MOI) of 10 in a 5% CO₂ incubator at 34°C for 8 h and then fixed with 4% paraformaldehyde and subjected to IF staining.

Chen J., Yang X., Si H., Gong Q., Que T., Li J., Li Y., Wu C., Zhang W., Chen Y., Luo Y., Zhu Y., Li B., Luo D., Hu B., Lin H., Jiang R., Jiang T., Li Q., Liu M., Xie S., Su J., Zheng X., Li A., Yao Y., Yang Y., Chen P., Wu A., He M., Lin X., Tong Y., Hu Y., Shi Z.L, & Zhou P. (2023). A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry. Cell, 186(4), 850-863.e16.

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Analyzing Allergen-Induced Cellular Responses

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Alternaria alternata and house dust mite (HDM; Dermatophagoides farina) extracts were obtained from Greer Laboratories (Lenoir, NC). Hydrogen peroxide solution, l-glutathione (GSH), N-acetyl-l-cysteine (NAC), PAMAM dendrimer, and mouse genomic DNA were purchased from Sigma-Aldrich (St Louis, Mo). AZ 10417808 (AQZ-1), PD 150606, and decanoyl-RVKR-CMK were procured from Tocris Bioscience (Bristol, United Kingdom). Hanks balanced salt solution (HBSS), acetoxymethyl ester form of fura-2 (fura-2 AM), SYBR Gold, YoYo-1, SYTOX Green, 4′,6-diamidino-2-phenylindole (DAPI), and EDTA were obtained from Thermo Fisher Scientific (Waltham, Mass). Etoposide was acquired from Abcam (Cambridge, United Kingdom). Naphthofluroscein, caspase-3, and calpain small interfering RNA (siRNA), control siRNA (fluorescein conjugate), siRNA transfection reagent, and media were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Fluorescently labeled antibodies to CD3 (145-2C11), CD25 (PC51), CD44 (IM7), CD16/CD32 (2.4G2), CD14 (rmC5-3), CD45R/B220 (RA3-6B2), and IgG₂a isotype control were purchased from BD Biosciences (San Jose, Calif).

Lee C.G., Soares V.D., Newberger C., Manova K., Lacy E, & Hurwitz J. (1998). RNA helicase A is essential for normal gastrulation. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13709-13713.

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Furin Inhibitor Impacts Transfection

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24 hrs after transfection, the furin inhibitor decanoyl-RVKR-CMK (Tocris) was added to the medium at final concentrations of 20uM, 50uM or 100uM. Cells were collected 72 hrs after transfection and lysed with RIPA buffer (Sigma) containing 1X Halt Protease Inhibitor (Thermo Scientific). The cell lysates were used for immunoprecipitation and western blotting experiments.

Zhang L., Mann M., Syed Z., Reynolds H.M., Tian E., Samara N.L., Zeldin D.C., Tabak L.A, & Ten Hagen K.G. (2021). Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation. bioRxiv.

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Furin-Mediated Cleavage of PTH(−6 to +34)

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[S1]PTH(−6 to +34) or [P1]PTH(−6 to +34) (10 µg/10 µL 10 mM acetic acid) was incubated with furin (8U/4 μL) (New England Biolabs) and furin buffer (50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)–KOH (potassium hydroxide) (pH 7.4), 150 mM NaCl, 5 mM MgCl₂, 16 mM imidazole, 2% glycerol, 0.15 mM EGTA, 500 μM ATP, 0.5% Triton-X100, 2 mM CaCl₂, and 2 mM β-mercaptoethanol) (RT, 60 min; final volume 30 µL) (New England Biolabs), as recommended by the manufacturer, before adding EGTA (100 μM, 3 μL). In some experiments, 1 μL of the reaction buffer was replaced with the furin inhibitor Decanoyl-RVKR-CMK (Tocris Bioscience; 50 μM in water). To assess cleavage, 5 µL of furin-treated or untreated peptides (1.5 µg each) was loaded onto a C18 column (2.1 mm × 150 mm, Higgins Analytical, Inc.), and a gradient of 4 to 76% ACN/0.05% TFA over 40 min (200 µL/min) was applied. To determine the molecular weight, 10 µL of furin-treated or untreated peptides was loaded onto a C18 ZipTip (Millipore Corporation) that had been rinsed with 100% ACN/0.01% TFA (2 × 20 μL) followed by equilibration with H₂O/0.01% TFA (5 × 20 μL). Peptide elution with 100% ACN/0.01% TFA was followed by speed vac and MassSpec analysis.

Hanna P., Khatri A., Choi S., Brabant S., Gild M.L., Piketty M.L., Francou B., Prié D., Potts JT J.r., Clifton-Bligh R.J., Linglart A., Gardella T.J, & Jüppner H. (2023). Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone. Proceedings of the National Academy of Sciences of the United States of America, 120(8), e2208047120.

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