4egi 1

Manufactured by Bio-Techne

Sourced in United Kingdom

4EGI-1 is a small molecule inhibitor that acts as a potent and selective inhibitor of the eIF4E-eIF4G interaction. It disrupts the eIF4F complex formation, thereby inhibiting cap-dependent translation initiation.

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Lab products found in correlation

Pf 4708671, by Bio-Techne (2 mentions) Rapamycin, by Bio-Techne (2 mentions) Dimethyl sulfoxide (dmso), by Merck Group (2 mentions) Rapamycin, by Cayman Chemical (1 mentions) Easysep magnet, by STEMCELL (1 mentions) Chir99021, by Bio-Techne (1 mentions) Chir99021, by Cayman Chemical (1 mentions) Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions) Macs cell separation kit, by Miltenyi Biotec (1 mentions) Penicillin, by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) X vivo 15, by Lonza (1 mentions) Torin1, by Bio-Techne (1 mentions) Nanobit ppi starter system, by Promega (1 mentions) Pcmv6 mammalian expression vector, by OriGene (1 mentions) Pcdna3 rluc polires fluc, by Addgene (1 mentions) 4e1rcat, by Bio-Techne (1 mentions) On targetplus non targeting pool d 001810 10 05, by Horizon Discovery (1 mentions) Pp242, by Bio-Techne (1 mentions) Rapamycin, by Selleck Chemicals (1 mentions)

6 protocols using 4egi 1

Mouse Hematopoietic Stem Cell Culture

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HSCs and HSPCs were cultured as described previously [28 (link)]. Briefly, mouse bone marrow cells were harvested and distributed into a single-cell suspension by gently drawing through a 22-gauge needle. Red blood cells were lysed in ammonium chloride–potassium (ACK) buffer. Lineage-depleted bone marrow cells were isolated by incubating whole bone marrow with biotin-conjugated lineage marker antibodies indicated above followed by incubation with Dynabeads M-280 Streptavidin (ThermoFisher) and separation with the EasySep magnet (Stem Cell Technologies). c-Kit⁺ cells were purified with the MACS cell separation kit (Miltenyi Biotec). Isolated HSCs and HSPCs were cultured in X-VIVO 15 (BioWhittaker) supplemented with 1% penicillin and streptomycin (Sigma). CHIR99021 and rapamycin (Cayman Chemical) reconstituted in DMSO were added to final concentrations of 3 μM (CHIR99021) and 5 nM (rapamycin) for all experiments unless otherwise indicated. For inhibition of S6K and eIF4E, cells were cultured in 3 μM CHIR99021 combined with 10 μM PF-4708671 (Tocris Bioscience) reconstituted in ethanol, 50 μM 4EGI-1 (Tocris Bioscience) reconstituted in DMSO, or both. Sorted cells were distributed into 96-well plates with 100 μl medium. One-half volume of medium and full drug volume were replaced every other day.

Nguyen-McCarty M, & Klein P.S. (2017). Autophagy is a signature of a signaling network that maintains hematopoietic stem cells. PLoS ONE, 12(5), e0177054.

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Characterizing eIF4E-eIF4G and 4EBP1 Interactions

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All eIF4E, eIF4G (including the Y624A, L629A and L630A binding mutant) and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G^604–646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3× FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene. PP242, Torin1, Rapamycin, 4EGi-1 and 4E1RCat were purchased from Tocris Bioscience, whilst all other chemicals unless otherwise stated were purchased from Selleck Chemicals. siRNAs targeting either 4EBP1 (ON-TargetPlus Human EI4EBP1, J-003005-12-0005) or non-targeting control (ON-TargetPlus Non-targeting pool, D-001810-10-05) were purchased from DHARMACON.

Frosi Y., Usher R., Lian D.T., Lane D.P, & Brown C.J. (2019). Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly. BMC Biology, 17, 40.

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Regulation of MAP2B Protein Localization

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PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl) was purchased from AccuStandard, (Lot #010610KS, New Haven, CT). All PCB 95 stock solutions were made in dry sterile dimethylsulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO). Plasmid encoding microtubule-associated-protein-2B (MAP2B)-pCAG-fusion protein red (FusRed) or MAP2B fused to enhanced green fluorescent protein (MAP2B-pCAG-EGFP) were generously provided by Dr. Gary Wayman (University of Washington, Pullman, WA) and have been previously characterized (Wayman et al. 2006 (link)). Predesigned Ambion Silencer Select siRNAs targeting mTOR (s132719), raptor (s143003), rictor (s160195), or scrambled siRNA (#4390844) were purchased from Thermo Fisher Scientific (Waltham, MA). FK506 was purchased from Cayman Chemical (Ann Arbor, MI); 4EGI-1, from Tocris (Minneapolis, MN); and rapamycin, from Selleckchem (Houston, TX).

Keil K.P., Miller G.W., Chen H., Sethi S., Schmuck M.R., Dhakal K., Kim J.W, & Lein P.J. (2018). PCB 95 Promotes Dendritic Growth in Primary Rat Hippocampal Neurons via mTOR-Dependent Mechanisms. Archives of toxicology, 92(10), 3163-3173.

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Compound Potency Evaluation by Dose-Response Assays

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Compound potency was assessed by dose-response assays in 96-well, flat-bottom microtiter plates (Sarstedt) or in 384-well, flat-bottom microtiter plates (Corning) as previously described (67 (link)). Plates were incubated at 30°C for the indicated time period. Growth was quantified by measuring OD₆₀₀, and the results were corrected for background media. All strains were assessed in biological-duplicate experiments with technical duplicates. Growth was normalized to the levels seen with the untreated controls, and data were plotted as a heat map using Java TreeView 1.1.6r4.
Dose-response matrixes (checkerboard assays) were performed in a similar manner except that the titers of 2-fold serial dilutions of compound 1 (indicated along the x axis of the checkerboard) and compound 2 (indicated along the y axis) were determined. Values representing the fractional inhibitory concentration index at 90% growth inhibition (FICI₉₀) were calculated as previously described (68 (link)).
BU-CMD hit compounds were newly supplied and dissolved in DMSO, 4EGI-1 (Tocris Biosciences) and rapamycin (BioShop) were dissolved in DMSO, fluconazole (Sequoia Research Products) was dissolved in sterile double-distilled water (ddH₂O), and calcium chloride (BioShop) was used to supplement yeast nitrogen base (YNB) media, as indicated.

Iyer K.R., Whitesell L., Porco JA J.r., Henkel T., Brown L.E., Robbins N, & Cowen L.E. (2020). Translation Inhibition by Rocaglates Activates a Species-Specific Cell Death Program in the Emerging Fungal Pathogen Candida auris. mBio, 11(2), e03329-19.

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Establishing an in vitro model of neurodegeneration

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Amyloid β protein fragment 1-42 (Aβ_1-42; Sigma-Aldrich, Louis, USA), rapamycin (Tocris Cookson, Bristol, UK), [2-((4-(5-ethylpyrimidin-4-yl)piperazine-1-yl) methyl)-5-(trifluoromethyl)-1H-benzo[d]imidazole] (PF4708671, Tocris Cookson), 2-[(4-(3,4-dichlorophenyl)-thiazol-2-ylhydrazono)-3-(2-nitrophenyl)]propionic acid (4EGI-1, Tocris Cookson) and NBQX (Tocris Cookson) were dissolved in dimethyl sulphoxide (DMSO). Kainate (Sigma-Aldrich), bicuculline methiodide (Tocris Cookson), and D-APV (Tocris Cookson) were dissolved in water. All drugs were stored in the freezer at 1,000-10,000 times the working solution, diluted immediately before use, and applied to the aCSF.

Wang Y.L., Wang J.G., Guo S., Guo F.L., Liu E.J., Yang X., Feng B., Wang J.Z., Vreugdenhil M, & Lu C.B. (2023). Oligomeric β-Amyloid Suppresses Hippocampal γ-Oscillations through Activation of the mTOR/S6K1 Pathway. Aging and Disease, 14(4), 1390-1406.

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Inhibition of eIF4F Complex Formation

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The eIF4F complex-formation inhibitor 4EGI-1 was obtained from Tocris Biosciences (Avonmouth, Bristol, UK) (CAS No. 315706-13-9/0). The compound was suspended in dimethyl sulfoxide (DMSO; Sigma) to make a 1000X stock at a concentration of 100 mM and diluted to final concentrations in DMEM (Gibco). Fresh solutions of the compound were prepared for individual experiments. A DMSO vehicle control was included in all studies.

Shives K.D., Massey A.R., May N.A., Morrison T.E, & Beckham J.D. (2016). 4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression. Viruses, 8(10), 287.

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