Pbabe puro braf v600e

Manufactured by Addgene

Sourced in United States

The PBabe-Puro-BRAF-V600E is a plasmid that expresses the BRAF-V600E mutant protein. It is a tool used in research to study the effects of the BRAF-V600E mutation.

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Lab products found in correlation

Polybrene, by Merck Group (2 mentions) Plx4720, by Selleck Chemicals (1 mentions) U0126, by Cell Signaling Technology (1 mentions) Mg132, by Cayman Chemical (1 mentions) Plx302, by Addgene (1 mentions) Pcmv vsv g, by Thermo Fisher Scientific (1 mentions) Egfr l858r, by Addgene (1 mentions) Egfr wt, by Addgene (1 mentions) Pcmv vsv g, by Addgene (1 mentions) Pcmv dr8.2 dvpr, by Addgene (1 mentions) Pbabe puro, by Addgene (1 mentions) 293ft cell, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Lr clonase, by Thermo Fisher Scientific (1 mentions) Pbabe puro hras v12, by Addgene (1 mentions) Pbabe puro hras v12 s35, by Addgene (1 mentions) Mycoalert mycoplasma detection kit, by Lonza (1 mentions) Sk mel 28, by American Type Culture Collection (1 mentions) Sk mel 2, by American Type Culture Collection (1 mentions) Pbabe puro mek dd, by Addgene (1 mentions)

6 protocols using pbabe puro braf v600e

Establishing BRAF(V600E) and MAFG Expressing Cell Lines

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RKO, SW48, VACO432 and DLD-1 cells were obtained from ATCC and grown as recommended by the supplier. For drug treatments in Figures 3A and 3B, RKO cells were treated with 0.1, 1 or 5 µM PLX4720 (Selleckchem), or 0.2, 2 or 10 µM U0126 (Cell Signaling Technology) for 24 hr. For Figure 3H, RKO cells were treated with 0.01, 1, 2, 4 or 8 µM MG132 (Cayman Chemical) for 4 hr. For Figure 3M, RKO cells were treated with 1 µM PLX4720 or 1 µM U0126 for 24 hr.
PFFs (ATCC) stably expressing BRAF(V600E) or MAFG were derived as follows. BRAF(V600E) cDNA (from plasmid pBabe-Puro-BRAF-V600E; plasmid 15269, Addgene) and MAFG cDNA (pMT2-MAFG (Blank et al., 1997 (link)); kindly provided by Volker Blank, McGill University/Lady Davis Institute for Medical Research) were cloned into pGIPZ-CMV (Open Biosystems), and the lentiviruses were transduced into PFFs and selected with puromycin for 24 hr.

Fang M., Ou J., Hutchinson L, & Green M.R. (2014). The BRAF Oncoprotein Functions Through the Transcriptional Repressor MAFG to Mediate the CpG Island Methylator Phenotype. Molecular cell, 55(6), 904-915.

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Generating and Transducing Lentiviral Plasmids

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A CRAF plasmid (pDONR223-CRAF), KRAS V12 plasmid (pDONR223-K-RAS V12), and BRAF V600E plasmid (pBabe-Puro-BRAF-V600E) were obtained from Addgene (Cambridge, MA). BRAF V600E was PCR amplified and cloned into a Gateway-system entry plasmid (pDonr207) (life, Carlsbad, CA). CRAF was transferred into the pDonr207 plasmid and a stop codon was added by a similar procedure. The following CRAF mutations were generated by overlap extension PCR and re-cloning into the pDonr207 plasmid by BP recombination: CRAF R391W, CRAF R391W R401H, and CRAF E478K. These entry clones were transferred into the following destination vectors: pDS_MYC and pDS_HA (n-terminal epitope tags; Alliance for Cellular Signaling), pDS-FB-hyg (retroviral expression vector, Alliance for Cellular Signaling), and pCEMM-CTAP-GWY (retroviral expression vector; GFP marker; tag not expressed due to presence of stop codon)46 (link). Retroviral particles were produced as described previously47 (link). Virus supernatants were harvested after 48 h and filtered. For the transduction, the cells were incubated with the viral supernatant and 4 μg/ml polybrene.

Atefi M., Titz B., Tsoi J., Avramis E., Le A., Ng C., Lomova A., Lassen A., Friedman M., Chmielowski B., Ribas A, & Graeber T.G. (2016). CRAF R391W is a melanoma driver oncogene. Scientific Reports, 6, 27454.

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Lentiviral and Retroviral Transduction in Breast Cancer Cells

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Plasmids including pCMV-VSV-G (#8454), pCMV-dR8.2 dvpr (#8455), pDONR223-MEKDD (#31202), pDONR223-H-RAS V12 (#31201), pLX302 (#25896), pBabe-puro-BRAF-V600E (#15269), EGFR L858R (#11012), EGFR WT (#11011) and pBabe-puro (#1764) were obtained from Addgene (Cambridge, MA, USA). Lentiviral donor plasmids were removed into the destination vector pLX302 using LR Clonase (Invitrogen, Carisbad, CA, USA). Recombined plasmids were verified by sequencing (Sangon Biotech, Shanghai, China). The infectious lentiviral particles were generated by co-transfecting pLX302 containing gene of interest, pCMV-VSV-G and pCMV-dR8.2 dvpr (at a 5:1:4 ratio) into the 293FT cells (Invitrogen, Carisbad, CA, USA). Retroviral particles were produced by transfection of Phenix293 cells (ATCC, Manassas, VA, USA) with pBabe containing gene of interest. Transfections were carried out using Lipofectamine 2000 according to the manufacture's instruction (Invitrogen, Carisbad, CA, USA). Breast cancer cells were infected with viruses in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After 48 h, stable transfected cells were selected with puromycin (2 μg/mL for T47D cells, 1 μg/mL for MCF-7 cells) until control plates became cleared at 3 days post-treatment.

Xu Y.C., Wang X., Chen Y., Chen S.M., Yang X.Y., Sun Y.M., Geng M.Y., Ding J, & Meng L.H. (2017). Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer. Theranostics, 7(4), 974-986.

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Melanoma Cell Lines Characterization

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SKMEL-2, SKMEL-28, MeWo, and A375 cells were purchased from American Type Culture Collection (ATCC) and grown as recommended. Neonatal primary human melanocytes were purchased from Life Technologies and grown as recommended by the supplier. All short-term melanoma cultures (YUGASP, YUHEF, YUTOGS, and YUVON) were obtained from the Yale SPORE in Skin Cancer, Yale University, and grown as recommended. SKMEL-103 and M318 cells were provided by Dr. Keiran Smally (Moffitt Cancer Center, Florida). MEL-ST cells were provided by Prof. Robert Weinberg (Whitehead Institute, MIT). All the cell lines were authenticated using STR analysis and tested for mycoplasma regularly using a MycoAlert Mycoplasma detection kit (Lonza, Allendale, NJ). Human IFI6 open reading frame was cloned in pcDNA3.1/hygro and used for rescue experiments. The plasmids pBabe puro-HRAS V12 (plasmid #15269), pBabe puro-HRAS V12 S35 (plasmid# 12274), pBabe puro-MEK-DD (plasmid #15268), and pBabe-puro-BRAFV600E (plasmid #15269) were purchased from Addgene. FG12 was a kind gift of Prof. David Baltimore, and FG12/NRASQ61K was a kind gift of Maria Soengas (CNIO, Spain).

Gupta R., Forloni M., Bisserier M., Dogra S.K., Yang Q, & Wajapeyee N. (2016). Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth. eLife, 5, e16432.

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Ectopic Expression of Key Signaling Proteins

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Plasmid 9018: 1271 pBabe puroL Myr HA Akt2, Plasmid 12523: pBabe puro Myr HA PIK3CA, Plasmid 12525: pBabe puro HA PIK3CA E545K, and Plasmid 15269: pBabe-Puro-BRAF-V600E were purchased from Addgene (Cambridge, MA). Transfection of HUVECs was done in six-well plates. Transfection reagents were mixed, vortexed, and incubated at room temperature for 30 min. Reagents (per six-well) were 95 μl of Opti-MEM Reduced Serum Medium with GlutaMAX (#51985-034; Invitrogen, Waltham, MA), 2 μl of siRNA (60 nM final concentration), and 4 μl of HiPerFect Transfection Reagent (#301705; Qiagen, Venlo, Netherlands). Cells were split and seeded at a density of 30–40% confluency in 650 μl of complete EGM-2 medium. Transfection reagents were added dropwise, and the cells were incubated overnight. The next day, 700 μl of EGM-2 was added (transfection reagents were not removed), and cells were incubated for additional 48 h. Transfected HUVECs were then split and seeded out in coculture with PaSMCs, as described in the following section.

Hellesøy M, & Lorens J.B. (2015). Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis. Molecular Biology of the Cell, 26(14), 2698-2711.

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Melanoma Cell Genetic Manipulation

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Melanoma cells were infected with the lentiviral expression system for short hairpin RNA (shRNA)-mediated BRAF and Usp5 knockdown and their control; pGIPZ Control, pGIPZ-USP5, and pGIPZ-BRAF were obtained from Open Biosystems. Knockdown of p53 was achieved with the following sense targeting sequence: p53; 5′-GACTCCAGTGGTAATCTAC-3′ cloned into pRetrosuperpuro (Oligoengine, Seattle, WA, USA). pBabe-puro-BRAF-V600E and pDEST-LTR-N-FLAG-HA-IRES-USP5 expression vectors was obtained from Addgene. HEK293T cells were transfected with the lentiviral packaging vectors pMD2.G and psPax2 (Addgene) together with the shRNA vectors to produce virus using PolyFect as described by the manufacturer (QIAGEN). The medium was changed to DMEM with 10% fetal bovine serum and after 48 hours, and the viral supernatant was collected. Viral supernatant containing 4 μg/mL of Polybrene (Sigma-Aldrich) was added to each melanoma cell line. After puromycin selection, Usp5 stable knockdown, overexpressing or control cells were used for analysis.

Potu H., Peterson L.F., Pal A., Verhaegen M., Cao J., Talpaz M, & Donato N.J. (2014). Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway. Oncotarget, 5(14), 5559-5569.

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