M51 super 8 fopflash

Manufactured by Addgene

Sourced in United States

The M51 Super 8× FOPFlash is a lab equipment product. It functions as a device for performing fluorescence-based assays.

Automatically generated - may contain errors

Lab products found in correlation

M50 super 8 topflash, by Addgene (7 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Lenti vpak packaging kit, by OriGene (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) Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions) Dual luciferase assay system, by Promega (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Prl tk, by Promega (1 mentions) Flustar omega, by BMG Labtech (1 mentions) Dual luciferase reporter system, by Promega (1 mentions) Dual luciferase reporter kit, by Promega (1 mentions) Microplate reader, by Tecan (1 mentions) M50 super 8x topflash, by Addgene (1 mentions) Cignal reporter assay, by Qiagen (1 mentions) Tgf β1, by Thermo Fisher Scientific (1 mentions)

9 protocols using m51 super 8 fopflash

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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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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Dual-Luciferase Assay for Wnt Signaling

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1.25 × 10⁵ cells/well were seeded in a 24 well plate. The following day, firefly luciferase constructs M50 Super 8× TOPFLASH (Addgene 12456) with 7 LEF1/β-catenin binding sites or the M51 Super 8× FOPFLASH (Addgene 12457) with 6 mutated LEF1/β-catenin binding sites were transfected at 7:1 ratio to a control renilla luciferase plasmid. For transfection, 1 μL polyethylenimine (PEI, 2 mg/mL) and 500 ng of total DNA were mixed with 600 μL of KSFM and incubated at room temperature for 10 minutes. Cells were transfected overnight. Six hours before harvest, 20 mM of lithium chloride (LiCl) or control sodium chloride (NaCl) was added. Lysates were collected with the Dual-Luciferase Reporter System (Promega) per the manufacturer's instructions. Firefly luciferase and renilla luciferase activity were assayed on a FLUstar Omega (BMG Labtech) plate reader sequentially for 10 seconds each. Firefly luciferase values were normalized to renilla luciferase as a transfection efficiency control.

Birdwell C.E., Prasai K., Dykes S., Jia Y., Munroe T.G., Bienkowska-Haba M, & Scott R.S. (2018). Epstein-Barr virus stably confers an invasive phenotype to epithelial cells through reprogramming of the WNT pathway. Oncotarget, 9(12), 10417-10435.

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Wnt Signaling Pathway Luciferase Assay

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A total of 1 × 10⁴ DLD1 cells were seeded on a white-bottom 96-well plate 20–24 h prior to transfection. On the day of transfection, each well received the following plasmids: M50 Super 8× TOPFlash plasmid (Addgene plasmid # 12456) or M51 Super 8× FOPFlash (TOPFlash mutant; Addgene plasmid # 12457), pCMV-Renilla^{29 (link)}, and pcDNA3-SnP_7 or pcDNA3-SnP_8. A total of 100 ng of plasmid DNA in a ratio of TOPFlash/FOPFlash : Renilla : SnP_7/SnP_8 uAb = 1:0.1:3 was mixed with Lipofectamine 3000 reagent in serum free Opti-MEM medium and added dropwise to each well after incubation at room temperature for 15 min. After 48 h of incubation, cells were lysed and the firefly and Renilla luminescence signals were measured sequentially by the dual-luciferase reporter kit (Promega). Plates were read on a microplate reader (Tecan). The luciferase activities were measured and normalized against the control Renilla activities.

Brixi G., Ye T., Hong L., Wang T., Monticello C., Lopez-Barbosa N., Vincoff S., Yudistyra V., Zhao L., Haarer E., Chen T., Pertsemlidis S., Palepu K., Bhat S., Christopher J., Li X., Liu T., Zhang S., Petersen L., DeLisa M.P, & Chatterjee P. (2023). SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders. Communications Biology, 6, 1081.

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Investigating HOTAIR's Role in Wnt Signaling

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To measure the effect of HOTAIR silencing on the canonical Wnt pathway, SF were transfected by electroporation (BTX) with the beta-catenin reporter M50 Super 8x TOPFlash (Addgene plasmid #12456) or M51 Super 8× FOPFlash, which contains mutated binding sites upstream of the luciferase reporter (Addgene plasmid #12457)^{91 (link)}. Both plasmids were a gift from Randall Moon. For normalization, pRenilla Luciferase Control Reporter Vectors (Promega) were co-transfected. 24 h after transfection, cells were transfected with GapmeR for HOTAIR or control as mentioned above. Luciferase activity was measured with a dual luciferase reporter assay system (Promega), and the results were normalized to the activity of Renilla luciferase. Wnt signaling activation was also determined by LEF (lymphoid enchancer factor) Cignal Reporter assay (Qiagen). SF were transfected with the WNT reporter, negative control or positive control (GFP) plasmid, respectively using nucleofection (BTX, 0.2 cm cuvette, 180 V, 20mS). Each plasmid was co-transfected with GapmeR for HOTAIR or control. After 48 h, luciferase activity was measure with the dual luciferase reporter assay system (Promega).

Elhai M., Micheroli R., Houtman M., Mirrahimi M., Moser L., Pauli C., Bürki K., Laimbacher A., Kania G., Klein K., Schätzle P., Frank Bertoncelj M., Edalat S.G., Keusch L., Khmelevskaya A., Toitou M., Geiss C., Rauer T., Sakkou M., Kollias G., Armaka M., Distler O, & Ospelt C. (2023). The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis. Nature Communications, 14, 8172.

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Hepatocellular Carcinoma Cell Culture

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For both 2D and 3D culture, all cell lines were cultured with the specific media that has been established for each cell line. HuH1, HuH7 and MHCC97-H 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). HepG2, Hep3B and SK-Hep-1 cells were cultured in Minimum Essential Medium (Life Technologies) supplemented with 10% FBS, PSG, non-essential amino acids and sodium pyruvate. M50 Super 8× TOPFlash and M51 Super 8× FOPFlash were purchased from Addgene (Cambridge, MA). pLKO.1-EpCAM shRNA and pLKO.1-eGFP shRNA lentiviral vectors were purchased from GE Healthcare (Lafayette, CO). R980-M19-663, a lentiviral vector with CMV13 promoter-driven firefly luciferase and eGFP expression was purchased from Frederick National Laboratory for Cancer Research (Frederick, MD). Lentiviral transduction was performed using Trans-Lentiviral shRNA Packaging System (GE Healthcare) or Lenti-vpak packaging kit (Origene, Rockville, MD). Fiduxosin was purchased from Sigma-Aldrich (St. Louis. MO). AV-606 was kindly provided by Avalon Pharmaceuticals (Germantown, MD). TGF-β1 was purchased from Peprotech (Rocky Hill, NJ).

Takai A., Fako V., Dang H., Forgues M., Yu Z., Budhu A, & Wang X.W. (2016). Three-dimensional Organotypic Culture Models of Human Hepatocellular Carcinoma. Scientific Reports, 6, 21174.

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Molecular Mechanisms of Cellular Regulation

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The siRNAs, including those targeting KIF23, PRC1, β-catenin and Amer1, were designed and synthesized by the Shanghai Sangon Company. The following plasmid constructs were used: M50 Super 8×TOPFlash and M51 Super 8×FOPFlash were gifts from Randall Moon (Addgene); pEGFP-C1-MKLP1 was a gift from Masanori Mishima; Renilla luciferase-Pol III was a gift from Norbert Perrimon; and the EGFP fusion expression vector pEGFP-C1 was obtained from Clontech. The plasmids harboring the Amer1-FL, ΔM, ΔA, ΔMA, KIF23 shRNAs (sh951, sh703) and the respective control vectors were provided by Shanghai GeneChem Co., Ltd. (Shanghai, China). Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) was used for plasmid transfection.

Liu Y., Chen H., Dong P., Xie G., Zhou Y., Ma Y., Yuan X., Yang J., Han L., Chen L, & Shen L. (2020). KIF23 activated Wnt/β-catenin signaling pathway through direct interaction with Amer1 in gastric cancer. Aging (Albany NY), 12(9), 8372-8396.

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