O glcnac

Manufactured by Merck Group

Sourced in United States

O-GlcNAc is a lab equipment product that functions as a post-translational modification protein. It is involved in the addition of N-acetylglucosamine to serine and threonine residues of target proteins.

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Anti-O-GlcNAc (9 citations) Anti-O-GlcNAc (CTD110.6) (4 citations) O-GlcNAc cycling enzyme inhibitors (2 citations)

3 protocols using o glcnac

Modulating O-GlcNAc levels in cell lines

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CL1-5 cells, which were kindly provided by Dr. Pan-Chyr Yang, and A549 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium. Culture media were supplemented with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂. The cells were treated with 5 μM OGA inhibitor Thiamet G (TMG; cat. no. 13237, Cayman Chemical, Ann Abor, MI, USA) or 50 μM PUGNAc (A7229, Sigma-Aldrich, St. Louis, MO, USA), in the complete medium for 24 h to increase O-GlcNAc levels. To manipulate the hexosamine biosynthetic pathway (HBP), 10 mM Glucosamine (G1514, Sigma-Aldrich, St. Louis, MO, USA), or 40 μM 6-Diazo-5-oxo-L-norleucine (DON) (D2141, Sigma-Aldrich, St. Louis, MO, USA) were used in the complete medium for 24 h.

Lin C.H., Liao C.C., Chen M.Y, & Chou T.Y. (2021). Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis. International Journal of Molecular Sciences, 22(7), 3463.

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Multimodal Analysis of Intracellular Signaling

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AML12, Hep3B, Huh7, and HepG2 cells were extracted using NP-40 lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and protease inhibitor cocktail tablets (Roche, Mannheim, Germany). Proteins were separated by electrophoresis on 8–15% SDS-PAGE gels and transferred to a nitrocellulose membrane (GE Healthcare Life science). After blocking with 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies at 4 °C overnight. The extracts were subjected to Western blot using antibodies directed against O-GlcNAc (Sigma-Aldrich, St Louis, MO, USA), OGA, IL-1β (Abcam, Cambridge, UK), PARP (Invitrogen, Waltham, MA, USA), cleaved caspase-3, IRE1α, P-p65, p65, P-IKKα/β, IKKα/β, P-IκB (Cell Signaling Technology, Danvers, MA, USA), OGT, p53, p21, PERK, P-ATM, XBP, Bcl2, Bax, lamin, α-tubulin, and β-actin (Santa Cruz, Dallas, TX, USA). After extensive washing with TBS-T, blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 30 min. Finally, the protein bands were visualized using an ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences). Signals were quantified by ImageJ software.

Lee S.J, & Kwon O.S. (2020). O-GlcNAc Transferase Inhibitor Synergistically Enhances Doxorubicin-Induced Apoptosis in HepG2 Cells. Cancers, 12(11), 3154.

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Uterine Artery Protein Expression Analysis

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Proteins (30 μg), extracted from uterine arteries (pooled from
2–3 animals to each n) were homogenized in lysis buffer containing
protease inhibitors. They were separated by electrophoresis on a 12%
polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma-Aldrich).
Non-specific binding sites were blocked with 5% skim dry milk in Tris-buffered
saline solution with Tween-20 (TBS-T, pH 7.6) for 1 h, at 24°C. Membranes
were incubated with the primary antibodies [O-GlcNAc, 1:500
(Sigma-Aldrich); phospho-eNOS^Ser1177, 1:500 (Cell Signaling
Technology); Total-eNOS, 1:500 (Cell Signaling Technology); phospho- glycogen
synthase kinase 3 beta (GSK3β)^Ser09, 1:250 (Cell Signaling
Technology); phospho-GSK3β^Tyr216, 1:250 (BD Biosciences);
Total-GSK3β, 1:250 (Cell Signaling Technology); Total-Akt, 1:1000 (Cell
Signaling Technology); OGT, 1:1000 (Cell Signaling Technology) or
β-actin, 1:10000 (Sigma-Aldrich)], overnight, at 4°C. On the next
day, membranes were removed from primary antibodies and washed with TBS-T.
Membranes were treated with the respective secondary antibodies for 1 hour at
room temperature. The protein bands were visualized using an enhanced
chemiluminescence detection system. Results were normalized to the intensity of
β-actin protein and are expressed as arbitrary units.

Justina V.D., Priviero F., Rodrigues dos Passos R J.u.n.i.o.r., Webb R.C., Lima V.V, & Giachini F.R. (2020). O-GlcNAc impairs endothelial function in uterine arteries from virgin but not pregnant rats: the role of GSK3β. European journal of pharmacology, 880, 173133.

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