Ogt2115

Manufactured by Bio-Techne

Sourced in United Kingdom

OGT2115 is a laboratory instrument designed for the detection and analysis of O-GlcNAc-modified proteins. It is a versatile tool used in biochemical research to study post-translational modifications and their role in cellular processes.

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Lab products found in correlation

Opti mem, by Thermo Fisher Scientific (2 mentions) Ab6505, by Abcam (1 mentions) Sc 372, by Santa Cruz Biotechnology (1 mentions) Sc 25825, by Santa Cruz Biotechnology (1 mentions) Ldh cytotoxicity assay, by Thermo Fisher Scientific (1 mentions) Histone h3, by Cell Signaling Technology (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Sc 25778, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Xav939, by Bio-Techne (1 mentions) Neurobasal media, by Thermo Fisher Scientific (1 mentions) Protein g affinity column, by GE Healthcare (1 mentions) Mmp 9 inhibitor 1, by Santa Cruz Biotechnology (1 mentions) Sb 3ct, by Abcam (1 mentions) Thalidomide, by Abcam (1 mentions) Tranilast, by Abcam (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Anti human gapdh, by Cell Signaling Technology (1 mentions) Anti human cd63, by Abcam (1 mentions) It901, by Bio-Techne (1 mentions)

10 protocols using ogt2115

Continuous Heparanase Inhibitor Delivery in SAH

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Three hours after SAH/sham surgeries, the Alzet osmotic pumps (Model 1003D, Alzet, Cupertino, CA) were installed subcutaneously in the neck region to provide the animals either vehicle or heparanase inhibitor, OGT2115 (MW: 495.3, Tocris Bioscience, Cat. #2710). The method has been described previously in details [34 (link)]. Briefly, rats were placed in a stereotactic frame under isoflurane anesthesia. The skull was exposed and a small hole over the right lateral ventricle (8 mm caudal to bregma, 1.5 mm lateral to midline, and 4 mm beneath the skull surface) was made to allow intracerebroventricular drug delivery. An initial loading dose of OGT2115 [0.05 μg in 5 μl artificial cerebrospinal fluid (aCSF)] or 5 μl vehicle (aCSF) was injected slowly into the lateral ventricle, followed by implantation of an infusion cannula with attached osmotic pump. This provided a continuous drug delivery at a rate of 1 μL/h to maintain CSF concentration of OGT2115 at approximate 0.4 μM, which is its IC50 for heparanase inhibition [35 (link)]. The OGT2115 was administered continuously until animals were sacrificed at 48 h.

Changyaleket B., Chong Z.Z., Dull R.O., Nanegrungsunk D, & Xu H. (2017). Heparanase promotes neuroinflammatory response during subarachnoid hemorrhage in rats. Journal of Neuroinflammation, 14, 137.

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Immunofluorescence and Western Blot Analysis of HPSE

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HPSE antibody (AB-476, Advanced Targeting Systems, San Diego, CA) and Heparan Sulfate-FITC antibody (H1890-10, US Biological, Salem, MA) were used for immunofluorescence studies (1:100). The following antibodies and dilutions were used for western blot: HPSE 1:200 (sc-25825, Santa Cruz Biotechnology, Dallas, TX), HPSE 1:200 (16673-AP-1, Proteintech Group, Inc., Rosemont, IL), NF-κB p65 1:500 (sc-372, Santa Cruz Biotechnology), IRF3 1:500 (11904, Cell Signaling Technology, Danvers, MA), IRF7 1:500 (4920, Cell Signaling), gB 1:10,000 (ab6505, Abcam) Histone H3 1:1000 (4499, Cell Signaling), GAPDH 1:1000 (sc-25778, Santa Cruz Biotechnology). Human HPSE expression constructs WT-HPSE, GS3-HPSE, C-DOM, and TIMB were a gift of Dr. Israel Vlodavsky (Rappaport Institute, Haifa, Israel) (Fux et al., 2009 (link)). All in vitro overexpression transfections were performed using Lipofectamine-2000 transfection reagent (Life Technologies), according to the manufacturer’s specifications. OGT 2115 was purchased from Tocris Biosciences and has been previously described as a HPSE inhibitor (Courtney et al., 2005 (link)). OGT 2115 was used at 10 μM unless otherwise specified. LDH cytotoxicity assay (Thermo Fisher 88953) was performed according to the manufacturer’s specifications.

Agelidis A.M., Hadigal S.R., Jaishankar D, & Shukla D. (2017). Viral Activation of Heparanase Drives Pathogenesis of Herpes Simplex Virus-1. Cell reports, 20(2), 439-450.

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OGT2115 Administration Before Exposure

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OGT2115 (Tocris Bioscience, Bristol, UK) was dissolved in DMSO and diluted with sterile water containing 5% Tween 80 and 30% PEG400. The mice were injected subcutaneously with OGT2115 (15 mg/kg) or an equal amount of vehicle (sterile water containing 1% DMSO, 5% Tween 80, and 30% PEG400) 6 h prior to Cl₂ exposure or histone H4 injection.

Zhang Y., Xu F., Guan L., Chen M., Zhao Y., Guo L., Li X., Zheng Y., Gao A, & Li S. (2022). Histone H4 induces heparan sulfate degradation by activating heparanase in chlorine gas-induced acute respiratory distress syndrome. Respiratory Research, 23, 14.

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Ex Vivo Sciatic Nerve Degeneration Assay

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The ex vivo sciatic nerve degeneration assay was conducted as previously described^{48 (link)}. Briefly both sciatic nerves were dissected from adult SD rats and cut into four equal segments. The segments were cultured in 24 well plates with 500 µl of Neurobasal media (ThermoFisher Scientific) supplemented with B27 (ThermoFisher Scientific), for the indicated number of days. Triangularis sterni (TS) muscles were dissected from the rib cage of B6.Cg-Tg mice pinned out flat as previously described^{61 (link)} and maintained in Ringers solution at 4 °C for 19 hrs. The Hpse inhibitor OGT2115 (Tocris Bioscience) was used at 8 μM^{62 (link)}. There is believed to be only one enzymatically active Hpse in mammals, and OGT2115 is the only commercially available inhibitor, its precise mechanism of action is not known. XAV939 the β-catenin inhibitor was used (Tocris Biosciences) at 40 μM^{63 (link)}. Both inhibitors were dissolved in DMSO and all controls were treated with DMSO only. Inhibitors were immediately added to the excised sciatic nerves or TS muscle.

Whitehead M.J., McGonigal R., Willison H.J, & Barnett S.C. (2018). Heparanase attenuates axon degeneration following sciatic nerve transection. Scientific Reports, 8, 5219.

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Inhibition of MIF, HPA-1, and MMP-9 in HUVECs

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To inhibit MIF activity, HUVECs were cotreated for 24 h with NS1 and either 100 μM p425 (6,6ʹ-[(3,3-dimethoxy[1,1ʹ-biphenyl]-4,4ʹ-diyl)bis(azo)]bis[4-amino-5-hydroxy-1,3-napthalenedisulphonic acid] tetrasodium salt; Calbiochem, La Jolla, CA, USA) or 50 μM ISO-1 ((S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid; Calbiochem). In addition, a rabbit anti-MIF polyclonal antibody (10 μg/ml) was used in this study and was purified from recombinant MIF-immunized rabbit serum using a protein G affinity column (GE Healthcare), as previously described [26 (link)]. To inhibit HPA-1, OGT 2115 (Tocris Bioscience, Bristol, UK) was used at the indicated concentration. To inhibit MMP-9, SB-3CT (Abcam, Cambridge, UK) and MMP-9 inhibitor I (Santa Cruz, Dallas, TX, USA) were used at the indicated concentrations. Control mouse and rabbit IgGs were purchased from LeadGene Biomedical (Taiwan).

Chen H.R., Chao C.H., Liu C.C., Ho T.S., Tsai H.P., Perng G.C., Lin Y.S., Wang J.R, & Yeh T.M. (2018). Macrophage migration inhibitory factor is critical for dengue NS1-induced endothelial glycocalyx degradation and hyperpermeability. PLoS Pathogens, 14(4), e1007033.

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Angiogenesis Inhibitors Cell Culture

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Eagle’s Minimum Essential Medium (EMEM) basal cell culture medium, RPMI-1640 basal cell culture medium and foetal bovine serum (FBS) were purchased from Life Technologies Ltd. (Paisley, U.K.). All chemical reagents used as described in subsequent sections were of the highest quality available and were sourced from Sigma-Aldrich Company Ltd. (Dorset, U.K.). Combretastatin A4, thalidomide and tranilast were sourced from Abcam plc. (Cambridge, U.K.). OGT 2115 was sourced from Tocris Bioscience (Bristol, U.K.). All anti-angiogenic drug compounds were dissolved in dimethyl sulphoxide (DMSO) to produce stock solutions of 10mM. Stock solutions were diluted in either cell culture medium or the appropriate assay buffer in order to achieve the desired final concentrations used during each experimental procedure.

Quayle L.A., Pereira M.G., Scheper G., Wiltshire T., Peake R.E., Hussain I., Rea C.A, & Bates T.E. (2017). Anti-angiogenic drugs: direct anti-cancer agents with mitochondrial mechanisms of action. Oncotarget, 8(51), 88670-88688.

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Myeloma Cell Lines in Heparanase Research

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Human myeloma cell lines utilized were: CAG (obtained from Dr. Joshua Epstein), MM1.R (obtained from Dr. Steven Rosen) and RPMI-8226/DOX40 cells (obtained from Dr. William Dalton). All cell lines were expanded and frozen in multiple vials upon receipt. All experiments were carried out within six weeks of thawing cells and not passaged more than five times. Cells were routinely tested for mycoplasma and retested prior to in vivo experiments. Generation of CAG cells expressing a high level of heparanase (HPSE-high) has been described [13 (link)]. The level of heparanase activity in HPSE-high cells is comparable to levels detected in bone marrow of many myeloma patients [13 (link)] and thus represent a physiologically relevant model for studying heparanase function in myeloma. These cells also express luciferase, which enabled monitoring of their location and growth in vivo. Myeloma cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Antibodies to the various proteins used in this study included the following: anti-human heparanase polyclonal antibody 1453, a kind gift from Dr. Israel Vlodavsky [52 (link)], anti-human CD63 (abcam), and anti-human GAPDH (Cell Signaling). The heparanase inhibitor Roneparstat was provided by Leadiant Biosciences S.A. and heparanase inhibitor OGT2115 and NF-κB inhibitor IT-901 were from Tocris Biosciences.

Tripathi K., Ramani V.C., Bandari S.K., Amin R., Brown E.E., Ritchie J.P., Stewart M.D, & Sanderson R.D. (2019). Heparanase promotes myeloma stemness and in vivo tumorigenesis. Matrix biology : journal of the International Society for Matrix Biology, 88, 53-68.

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Matrigel Invasion Assay with Heparanase Inhibitor

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Matrigel invasion chamber (BD Biosciences, Heidelberg, Germany) assays were performed essentially as described before [15 (link)] in the presence and absence of 1 μM heparanase inhibitor OGT 2115 (2-[4-[[3-(4-Bromophenyl)-1-oxo-2-propenyl]amino]-3-fluorophenyl]-5-benzoxazoleacetic acid; Cat.no. 2710, TOCRIS bioscience, Wiesbaden-Nordenstadt), using an invasion time of 48 h. Relative invasiveness was expressed as percentage of cells on compound-treated compared to control inserts (n > 3).

El-Nadi M., Hassan H., Saleh M.E., Nassar E., Ismail Y.M., Amer M., Greve B., Götte M., El-Shinawi M, & Ibrahim S.A. (2020). Induction of heparanase via IL-10 correlates with a high infiltration of CD163+ M2-type tumor-associated macrophages in inflammatory breast carcinomas. Matrix Biology Plus, 6-7, 100030.

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Cell Cycle Distribution Analysis by Flow Cytometry

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Cell cycle distribution was monitored by flow cytometry analysis. Briefly, Untreated MCF-7 and MDA-MB-435 cells were used as negative controls. Cells were treated with 5-aza-dC alone or in combination with 1 µM OGT2115, a heparanase inhibitor (Tocris Bioscience, Bristol, UK), respectively [17] (link). Cells with or without treatment were trypsinized, washed with phosphate-buffered saline (PBS) and fixed with 75% ethanol overnight at −20°C. The cells were washed twice with 1×PBS and added 1 ml of propidium iodide (PI, Sigma, USA) staining solution (50 µg/ml) to cell pellet. Then, 50 µl of RNase A stock solution was added and incubated for 3 h at 4°C. The cell cycle was analyzed by a flow cytometry (Beckman Coulter, Inc., USA).

Jiao F., Bai S.Y., Ma Y., Yan Z.H., Yue Z., Yu Y., Wang X, & Wang J. (2014). DNA Methylation of Heparanase Promoter Influences Its Expression and Associated with the Progression of Human Breast Cancer. PLoS ONE, 9(3), e92190.

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Quantifying HSV-1 Plaque Reduction

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Monolayers of HCE cells were infected with HSV-1 (KOS MOI 0.1) in Opti-MEM (Thermo Fisher Scientific). After 2 h incubation at 37°C, 5% CO₂, inoculation solution was aspirated, cells were washed once with PBS, and complete MEM containing 10 μM DMSO or specified concentration of OGT 2115 (Tocris Biosciences) was added for 24 h. Culture supernatants were then collected, centrifuged at 13,000 rpm for 1 min, serially diluted in Opti-MEM, and overlayed on confluent monolayers of Vero cells in 24-well plates. After 2 h incubation, cells were washed, and complete DMEM containing 0.5% methylcellulose (Fisher Scientific) was added to cells for 48 to 72 h. To visualize and count plaques, cells were fixed with 100% methanol and stained with crystal violet solution.

Agelidis A.M., Hadigal S.R., Jaishankar D, & Shukla D. (2017). Viral Activation of Heparanase Drives Pathogenesis of Herpes Simplex Virus-1. Cell reports, 20(2), 439-450.

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