M50 super 8 topflash

Manufactured by Addgene

Sourced in United States

The M50 Super 8× TOPFlash is a lab equipment product. It is a fluorescent reporter system used for the analysis of Wnt/β-catenin signaling pathway activity.

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Lab products found in correlation

M51 super 8 fopflash, by Addgene (7 mentions) Dual luciferase reporter assay system, by Promega (5 mentions) Passive lysis buffer (plb), by Promega (3 mentions) M51 super 8 fopflash topflash mutant, by Addgene (3 mentions) Prl tk luc, by Promega (2 mentions) Lar 2, by Promega (2 mentions) Human beta catenin pcdna3, by Addgene (2 mentions) Pcdna3 s33y beta catenin, by Addgene (2 mentions) Geneart site directed mutagenesis system, by Thermo Fisher Scientific (2 mentions) Lenti vpak packaging kit, by OriGene (2 mentions) Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions) Plko 1 epcam shrna, by GE Healthcare (2 mentions) Plko 1 egfp shrna lentiviral vectors, by GE Healthcare (2 mentions) Trans lentiviral shrna packaging system, by GE Healthcare (2 mentions) Dual luciferase reporter dlr assay system, by Promega (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Pdl coated white 96 well cell culture plate with solid flat bottom, by Greiner (1 mentions) Stop glo, by Promega (1 mentions) Dual luciferase assay system, by Yeasen (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

TOPFlash (64 citations) Super 8× TopFlash (9 citations) M50 Super 8× TOPFlash plasmid (2 citations)

25 protocols using m50 super 8 topflash

Wnt Signaling Pathway Activation Assay

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6 × 10⁵ cells ml⁻¹ ΔFZD_1-10 HEK293 T cells were seeded onto PDL-coated white 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Next day, cells were transfected with 20 ng of SNAP-tagged receptor, 20 ng M50 Super 8× TOPFlash (Addgene, 12456), 2 ng pRL-TK Luc (Promega, E2241) and 58 ng pcDNA plasmid DNA to a final amount of 100 ng of plasmid DNA per well. Four hours after transfection, medium was changed to starvation medium (DMEM without FBS) containing either vehicle or 1000 ng/ml recombinant WNT-3A for FZD transfected cells or 100 nM SAG1.3 for SMO-transfected cells. Twenty-four hours after stimulation, cells were lysed gently shaking with 20 µl 1× Passive Lysis Buffer (Promega, E1910) for 15 min. Subsequently, 20 µl of LAR II (Promega, E1910) were added to all wells after which luminescence (580-80 nm) was read and then 20 µl of Stop & Glo (Promega, E1910) were added to all wells after which luminescence (480-80 nm) was read again with a CLARIOstar microplate reader (BMG). Data were analyzed using GraphPad Prism 6.

Turku A., Schihada H., Kozielewicz P., Bowin C.F, & Schulte G. (2021). Residue 6.43 defines receptor function in class F GPCRs. Nature Communications, 12, 3919.

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Cloning and Mutagenesis of Plasmid Constructs

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Mouse Fzd6 cDNA was cloned into a Cherry-C1 plasmid (K4-Fzd6-Cherry) and pEGFP-C1 Mouse Ceslr1 cDNA that was cloned into a pEGFP-N1 plasmid (pEGFPN1-Celsr1), obtained from Dr. Elaine Fuchs’s laboratory (Devenport and Fuchs 2008 (link)) (The Rockefeller University, New York, USA). pcDNA3.1+/C-eGFP-VANGL1 construct (clone ID: OHu10988) was purchased from the Genscript Company (Piscataway, NJ 08854, USA). Fzd6 p.Gln88Glu and VANGL1 p.Gly644Val mutations were introduced into their wild-type (WT) constructs through site-directed mutagenesis using GeneArt® Site-Directed Mutagenesis System (Thermo Fisher Scientific, CAT#: A14604). All plasmids were validated by sequencing analyses. M50 Super 8 × TOPFlash (Veeman et al. 2003a (link), b (link)), human beta-catenin pcDNA3, and pcDNA3-S33Y beta-catenin (Kolligs et al. 1999 (link)) were purchased from Addgene (https://www.addgene.org/ ). The pAP1-Luc reporter construct (Part#: 219074) was purchased from Agilent.

Tian T., Lei Y., Chen Y., Karki M., Jin L., Finnell R.H., Wang L, & Ren A. (2020). Somatic mutations in planar cell polarity genes in neural tissue from human fetuses with neural tube defects. Human genetics, 139(10), 1299-1314.

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Quantifying β-catenin Activity Modulation

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The TCF/LEF reported plasmid for β-catenin activity (M50 Super 8 × TOPFlash; plasmid#12456; Addgene, Cambridge, MA, United States) was a gift from Randall Moon (Veeman et al., 2003 (link)). For dual luciferase assay, cells were seeded into 12-well plates. The cells were transfected with 8 × TOPFlash plasmid and indicated siRNA. After transfection for 48 h, cells were co-cultured with H. pylori strain. The cell lysates were collected and subjected to a dual luciferase assay system (Yeasen Biotech, Shanghai, China) according to the manufacturer’s protocol.

Xu X., Shu C., Wu X., Ouyang Y., Cheng H., Zhou Y., Wang H., He C., Xie C., He X., Hong J., Lu N., Ge Z., Zhu Y, & Li N. (2022). A positive feedback loop of the TAZ/β-catenin axis promotes Helicobacter pylori-associated gastric carcinogenesis. Frontiers in Microbiology, 13, 1065462.

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Measuring Wnt Pathway Activation

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HT-29 cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturer’s instructions, using 250ng of the TOPFlash reporter gene construct (M50 Super 8× TOPFlash, Plasmid #12456, Addgene) and 500ng of pcDNA3-LUST and/or 500ng of pcDNA-β-catenin construct. Luciferase reporter gene expression was measured according to the manufacturer’s protocol (Dual-luciferase Reporter assay System, Promega). The luciferase activity was normalized to Renilla luciferase activity from co-transfected internal control plasmid pRL-CMV.

Di Cecilia S., Zhang F., Sancho A., Li S.D., Aguilo F., Sun Y., Rengasamy M., Zhang W., Vecchio L.D., Salvatore F, & Walsh M.J. (2016). RBM5-AS1 is critical for self-renewal of colon cancer stem-like cells. Cancer research, 76(19), 5615-5627.

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Molecular Mechanisms of Chicken Antiviral Signaling

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pCAGGS-myc-L was obtained from Shanhui Ren (Lanzhou Veterinary Research Institute). pcDNA-HA-NP, pcDNA-HA-P, and pRL-TK were obtained from Wenbin Wang (Shandong Academy of Agricultural Science). M50 Super 8× TOPFlash (catalog number 12456) and M51 Super 8× FOPFlash (catalog number 12457) were purchased from Addgene (USA). The chicken IFN-β-luc was preserved in our laboratory. The β-catenin, HSP90AA1, and HSP90AB1 genes of chicken were amplified and cloned into the pCAGGS plasmid with fused N-terminal tags (β-catenin, HA tag; HSP90AA1/HSP90AB1, Flag tag; HSP90AA1, HA tag). The primers that were used in this study are listed in Table 4.
The transfections were performed according to the protocol of the Turbofect transfection reagent (Thermo Fisher Scientific). For the plasmid transfections, 5 × 10⁵ DF-1 cells were seeded onto 12-well plates and were then transfected with the indicated amounts of plasmids. For siRNA transfection, 5 × 10⁵ DF-1 cells were seeded onto 12-well plates and then transfected with the siRNA at the concentration of 100 nM. The siRNAs for β-catenin, HSP90AA1, and HSP90AB1 were designed and synthesized by RiboBio (China). The siRNAs that were used in this study are listed in Table 5.

Wang C., Wang T., Hu R., Duan L., Hou Q., Han Y., Dai J., Wang W., Ren S., Liu H., Wang X., Xiao S., Li N., Wang J, & Yang Z. (2023). 9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3β and HSP90β. Journal of Virology, 97(3), e01984-22.

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Wnt/TCF Signaling Reporter Assay

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A reporter containing TCF/LEF elements (M50 Super 8 × TOPFlash) (Veeman et al., 2003 (link)) (Addgene, Cambridge, MA, United States) was used as the reporter vector (Harada et al., 2016 (link)). The luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, United States), as described previously (Yamashita et al., 2014 (link)).

Kubo C., Ogawa M., Uehara N, & Katakura Y. (2020). Fisetin Promotes Hair Growth by Augmenting TERT Expression. Frontiers in Cell and Developmental Biology, 8, 566617.

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Measuring β-catenin and EpCAM Transcriptional Activity

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Cells were co-transfected using Lipofectamine 2000 (Life Technologies) with 500 ng M50 Super 8× TOPFlash or M51 Super 8× FOPFlash (Addgene), and 50 ng pRL-null Renilla luciferase to measure β-catenin transcriptional activity. For EpCAM transcriptional activity, cells were co-transfected with 500 ng pGL3-EpCAM2.2 and 50 ng pRL-null Renilla luciferase. Cells were then treated with DMSO or 10 µM PMZ in triplicate for 24 hours. The Dual-Luciferase Reporter Assay System (Promega) was used to determine firefly and renilla luciferase activity according to the manufacturer's instructions.

Fako V., Yu Z., Henrich C.J., Ransom T., Budhu A.S, & Wang X.W. (2016). Inhibition of wnt/β-catenin Signaling in Hepatocellular Carcinoma by an Antipsychotic Drug Pimozide. International Journal of Biological Sciences, 12(7), 768-775.

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Lentiviral Transduction of Hepatocellular Carcinoma Cell Lines

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HUH7, HUH1 and MHCC97H cells were cultured in Dulbecco’s modified Eagle Medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine (PSG). Hep3B cells were cultured in Minimum Essential Medium (Life Technologies) supplemented with 10% FBS, PSG, non-essential amino acids and sodium pyruvate. pLKO.1-PMPCB shRNA, pLKO.1-EpCAM shRNA and pLKO.1-eGFP shRNA lentiviral vectors were purchased from GE Healthcare (Lafayette, CO). R980-M15–663, a lentiviral vector with CAG promoter-driven firefly luciferase and eGFP expression was purchased from the Protein Expression Laboratory, Frederick National Laboratory for Cancer Research (Frederick, MD). Lentivirus particles were generated using Trans-Lentiviral shRNA Packaging System (GE Healthcare) or Lenti-vpak packaging kit (Origene, Rockville, MD). pCMV6-PMPCB-DDK plasmid was purchased from Origene. M50 Super 8× TOPFlash, M51 Super 8× FOPFlash and pCI-neo beta catenin S33Y plasmid were purchased from Addgene (Cambridge, MA). InSolution^™ Caspase Inhibitor VI (Z-VAD), GSK-3 Inhibitor IX (BIO) and GSK-3 Inhibitor IX Control (MeBIO) were purchased from Millipore (Billerica, MA). All cell lines were tested for mycoplasma and authenticated via STR analysis (August, 2015). Cells were passaged less than 15X after first thaw from liquid nitrogen.

Takai A., Dang H., Oishi N., Khatib S., Martin S.P., Dominguez D.A., Luo J., Bagni R., Wu X., Powell K., Ye Q.H., Jia H., Qin L.X., Chen J., Mitchell G.A., Luo X., Thorgeirsson S.S, & Wang X.W. (2019). Genome-wide RNAi Screen identifies PMPCB as a therapeutic vulnerability in EpCAM+ hepatocellular carcinoma. Cancer research, 79(9), 2379-2391.

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Dual Luciferase Reporter Assay for TCF/LEF Activity

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The TCF/LEF reporter plasmid (M50 Super 8× TOPFlash, plasmid #12456; Addgene, Cambridge, MA, USA) and the mutant TCF/LEF reporter plasmid (M51 Super 8× FOPFlash, plasmid #12457; Addgene) were gifts from Dr. Randall T. Moon [41 ]. The Renilla luciferase control plasmid was purchased from Promega (Madison, WI, USA). Cells were transfected with 8× TOPFlash or 8× FOPFlash and other additional plasmids as indicated along with pRL-TK (Promega) to normalize the transfection efficiencies. After transfection for 48 h, cells were washed with PBS and lysed in passive lysis buffer (Promega). The lysates were subjected to a dual luciferase assay system (Promega) and processed according to the manufacturer’s protocol, followed by measurement using a Lumat LB-9501 luminometer (Berthold Analytical Instruments, Nashua, NH, USA). TOP/FOP ratios represented the mean of three independent experiments, performed in triplicate.

Gao J., Zhao C., Liu Q., Hou X., Li S., Xing X., Yang C, & Luo Y. (2018). Cyclin G2 suppresses Wnt/β-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1. Journal of Experimental & Clinical Cancer Research : CR, 37, 317.

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Luciferase Assay of miR-17-92 Promoters

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For the luciferase assay, we used M50 super 8× TOP flash (addgene) as a template. The wild-type and mutated promoter regions of miR-17-92 were amplified via PCR and replaced with the pre-existing promoter sequence. DNA fragments of miR-17-92 promoter #1, miR-17-92 promoter #2, miR-17-92 promoter #3, or the wild-type/mutated promoter region of c-Myc were used. pSV40 Renilla-luc was used as a negative control. The indicated combination of the reporter plasmids or a single plasmid was transfected into MEFs or HEK 293T cells. Following 48 h of incubation, transfectants were subjected to a luciferase reporter assay using PicaGene Dual Sea Pansy Luminescence kit (TOYO B-NET, Tokyo, Japan) according to the manufacturer’s protocol. Raw luciferase values were normalized by the value of mock-transfected cells.

Ishida T., Ueyama T., Ihara D., Harada Y., Nakagawa S., Saito K., Nakao S, & Kawamura T. (2023). c-Myc/microRNA-17-92 Axis Phase-Dependently Regulates PTEN and p21 Expression via ceRNA during Reprogramming to Mouse Pluripotent Stem Cells. Biomedicines, 11(6), 1737.

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