Pcw57.1 4ebp1 4xala

Manufactured by Addgene

The PCW57.1-4EBP1_4xAla is a plasmid designed for protein expression in E. coli. It contains the 4EBP1 gene with four alanine substitutions. The plasmid allows for the production of the modified 4EBP1 protein.

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5 protocols using pcw57.1 4ebp1 4xala

NFAT Luciferase Reporter Assay

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The CXCL8, Nluc and mCherry reporter vectors were generated as described in S1 Table. Positions 79 to 153 of NM_000584 was inserted immediately upstream of pNFAT Fluc (Addgene plasmid #10959 [58 (link)]) to form pNFAT 53’ Fluc. PCW57.1-4EBP1_4xAla (from Prof David Sabatini, Addgene plasmid # 38240 [4 (link)]); NFAT luciferase reporter (from Toren Finkel, Addgene plasmid #10959 [58 (link)]); pCNA-TAK1-WT, pCMV5-TAB1-FLAG, pCDNA-myr-AKT, pCDNA JNK/SAPK-MKK7 and pCMV-PKAca.

Ang Z., Koean R.A., Er J.Z., Lee L.T., Tam J.K., Guo H, & Ding J.L. (2019). Novel AU-rich proximal UTR sequences (APS) enhance CXCL8 synthesis upon the induction of rpS6 phosphorylation. PLoS Genetics, 15(4), e1008077.

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Establishing Inducible 4EBP1 Expression

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293GP cells were plated in 10cm plates and transfected using lipofectamine 2000 with 6μg retroviral and 1.5μg pMD2G plasmids. Supernatent was harvested from packaging cells on two consecutive days beginning 24hr following transfection. 8μg/ml polybrene was added to virus containing supernatant before adding to target cells. MCF7 parental cells were first infected with a CMV driven expression construct encoding rtTA3 (addgene:26730). Cells were selected in 250μg/ml hygromycin for 7 days. 293T cells were plated in 10cm plates and transfected using lipofectamine 2000 with 6μg lentiviral pCW57.1-4EBP1_4xAla (Addgene: 38240), 1.5μg pMD2G and 4.5μg psPax2. Virus containing supernatant with 8μg/ml polybrene was collected on two consecutive days and added to MCF7-rtTA3. Cells were selected for 3 days in 2μg/ml puromycin. Cells were thereafter maintained in DMEM F12 containing tetracycline-free FBS and 1% Penicillin/Streptomycin supplemented with 0.5ug/ml puromycin and 100ug/ml hygromycin.

Boyer J.A., Sharma M., Dorso M.A., Mai N., Amor C., Reiter J.M., Kannan R., Gadal S., Xu J., Miele M., Li Z., Chen X., Chang Q., Pareja F., Worland S., Warner D., Sperry S., Chiang G.G., Thompson P.A., Yang G., Ouerfelli O., de Stanchina E., Wendel H.G., Rosen E.Y., Chandarlapaty S, & Rosen N. (2024). eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer. bioRxiv.

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In Vitro and In Vivo Compound Preparation

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For in vitro studies MLN0128 (Selleckchem Houston, TX, USA) was dissolved in DMSO, phenfomin was dissolved in DMEM. For in vivo studies MLN0128 was dissolved in 1-methyl-2-pyrrolidinone (NMP), then diluted in 15% PEG diluted in water; phenformin was diluted in water at 1.8 mg/ml. Rapamycin was purchased from LC Laboratories (Woburn, MA) and dissolved in DMSO. Antibodies from Cell Signaling Technology (Beverly, MA, USA) used for immunoblots were diluted 1:1,000 and included phospho-p70 S6 kinase (Thr389 #9234), phospho-S6 (Ser235/236 #4858), total 4E-BP1 (#9644), phospho-4E-BP1 (Thr37/46 #2855), phospho-Akt (Ser473 #4060), phospho-Akt (Thr308 #4056), phospho-NDRG1 (Thr346 #5482), Phospho-Tuberin/TSC2 (Thr1462 #3611), phospho-GSKα/β (Ser21/9 #9331), beta-actin (#4970), cleaved PARP (Asp214 #5625), cleaved caspase 3 (Asp175 #9661) and phospho-Raptor (Ser792 #2083). Anti-Hif-1alpha (C-Term #10006421 1:200) antibody was purchased from Cayman Chemical and anti-GLUT1 (GT11-A 1:1,000) antibody was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Plasmid expressing dox-inducible 4EBP1 4Ala (pCW57.1-4EBP1_4xAla, plasmid # 38240) and control vector (pCW57.1, plasmid # 41393) were purchased from Addgene.

Momcilovic M., McMickle R., Abt E., Seki A., Simko S.A., Magyar C., Stout D.B., Fishbein M.C., Walser T.C., Dubinett S.M, & Shackelford D.B. (2015). Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers. Cancer research, 75(22), 4910-4922.

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Generation of Myr-AKT and 4EBP1-4A Cell Lines

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MCF-7 cells overexpressing a myristoylated form of AKT (Myr-AKT) were generated by transfection with 1 µg of pcDNA3-Myr-HA-AKT1 (Addgene plasmid #9008). Transfection was aided by pre-incubation with lipofectamine 2000 and used according to the manufacturer’s instructions. Media was changed 6 hours after transfection, and cells were harvested after an additional 24 hours. MDA-MB-468 and MCF-7 cells expressing inducible 4EBP1 T37A/T46A/S65A/T70A (4EBP1–4A) were generated by lentiviral transduction with pCW57.1–4EBP1_4xAla (Addgene plasmid #38240)⁴⁸. Lentivirus were packaged in 293T cells, and viral supernatant was filtered with 0.45-µm PVDF filters and incubated with target cells for six hours. Cells were cultured in virus-free media for two days, and infected cells were selected using Puromycin (2 μg/ml) for 3 days.

Lee B.J., Boyer J.A., Burnett G.L., Thottumkara A.P., Tibrewal N., Wilson S.L., Hsieh T., Marquez A., Lorenzana E.G., Evans J.W., Hulea L., Kiss G., Liu H., Lee D., Larsson O., McLaughlan S., Topisirovic I., Wang Z., Wang Z., Zhao Y., Wildes D., Aggen J.B., Singh M., Gill A.L., Smith J.A, & Rosen N. (2021). Selective Inhibitors of mTORC1 Activate 4EBP1 and Suppress Tumor Growth. Nature chemical biology, 17(10), 1065-1074.

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Dual Luciferase Reporter Plasmid Generation

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All plasmids for the Dual Luciferase Reporter were generated by subcloning gene body (GAPDH) or 5’UTR elements (ER, Myc) into Addgene 45642, pLenti CMV rtTA3 Hygro (w785-1) (Addgene: 26730), pCW57.1-4EBP1_4xAla (Addgene: 38240).

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