The Ca2+ sensitive transcriptional luciferase reporter pGL4.30[luc2P/NFAT-RE/Hygro] (Promega, E8481) was transiently transfected into the 5HT2BR stable cell line. Briefly, 3 × 106 cells were plated in a Nunc Cell Culture Treated flask (25 cm2, ThermoFisher) and transfected with lipofectamine 2000 (ThermoFisher) plus 1 µg of plasmid DNA according to the manufacturer’s protocol. The following day, culture media was replaced with induction media (DMEM + 10% dialyzed FBS supplemented with 1 µg/ml doxycycline), and 24 h later cells were re-plated into 96 well, solid white plates (Costar, 3917). After allowing 3 h for cells to adhere, drugs were added at 20× concentration. For antagonist experiments, cells were incubated with 5HT2B R antagonists for 2 h prior to subsequent addition of agonist. After 18 h culture in the presence of agonist, plates were assayed using the ONE-Glo Luciferase Assay System (Promega, E6120) and read on a GloMax-Multi Detection System plate reader (Promega).
Pgl4.30 luc2p nfat re hygro
Manufactured by Promega
Sourced in Japan, United States
The PGL4.30[luc2P/NFAT-RE/Hygro] is a reporter vector that contains the firefly luciferase gene (luc2P) under the control of the NFAT (Nuclear Factor of Activated T-cells) response element. It also includes a hygromycin resistance gene for selection purposes. This product is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Pef4 myc his b, by Thermo Fisher Scientific (2 mentions)
Prl tk plasmid, by Promega (2 mentions)
Dharmafect, by Horizon Discovery (2 mentions)
Synergy 2, by Agilent Technologies (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Glomax multi detection system plate reader, by Promega (1 mentions)
One glo luciferase assay system, by Promega (1 mentions)
Pgl3 promoter, by Promega (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Pge2 immunoassay kit, by R&D Systems (1 mentions)
Pgl4.32 luc2p nf κb re hygro, by Promega (1 mentions)
Il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Hdac1, by Cell Signaling Technology (1 mentions)
Hdac2, by Cell Signaling Technology (1 mentions)
Hdac6 flag, by Addgene (1 mentions)
Hdac1 flag, by Addgene (1 mentions)
11 protocols using pgl4.30 luc2p nfat re hygro
1
Transcriptional Luciferase Assay for 5HT2B Receptor
2
Quantification of NFAT Luciferase Activity
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Luciferase activities were quantified using pGL4.30 [luc2P/NFAT-RE/Hygro] (Promega, Tokyo, Japan), pRL-SV40 vectors, and the pGL3 promoter (Promega) for the NFAT response assay. The reporter assays proceeded using the Dual-Luciferase Reporter Assay System (Promega). The two reporter plasmids were co-transfected into fcwf-4 cells with or without 0.05 μg/mL phorbol 12-myristate-13-acetate (PMA; Sigma–Aldrich, Tokyo, Japan) and 0.142 μg/mL ionomycin (Sigma–Aldrich) to stimulate calcium signaling for each assay. Total cell lysates were prepared with reporter lysis buffer provided with the Dual-Luciferase Reporter Assay System at 48 h p.t. before the assay. Luciferase activities were quantified in triplicate assays using a Lumat LB9507 system (Berthold Technologies, Tokyo, Japan).
3
NFAT-driven Luciferase Assay for DYRK1A
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The firefly luciferase reporter assay was performed using a One-Glo Assay System (Promega) according to the manufacturer's instructions. The transcriptional activity of NFATc1 in 293T cells was measured with a reporter vector (pGL4.30 luc2P/NFAT-RE/Hygro) (Promega) that expresses the firefly luciferase gene under the control of an NFAT-RE promoter. 293T cells were transfected with the DYRK1A-expressing plasmid and reporter plasmid. On the next day, cells were treated with IM (2.5 μM) and phorbol-12-myristate-13-acetate (10 nM) along with the indicated amounts of inhibitors. Cells were further incubated for 8 h and then cell lysates were harvested to measure luciferase activity.
4
Luciferase Assay for NFAT Activation
5
HDAC Inhibition and Inflammation Regulation
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MPT0G009 and SAHA were synthesized by Dr. Jing-Ping Liou to >98% purity.12 The non-conjugated primary antibodies used were against HDAC6 or β-actin (Epitomics Inc., Burlingame, CA, USA), HDAC1, HDAC2, HDAC3 or histone H3 (Cell Signaling Technology, Danvers, MA, USA), or acetyl-histone H3 or p21/WAF1/Cip1 (Millipore, Temecula, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD51/61 antibodies was from BD Biosciences (San Jose, CA, USA). The labeled secondary antibodies were horseradish peroxidase- or FITC-conjugated anti-mouse or anti-rabbit IgG antibodies (Jackson ImmunoResearch Inc., West Grove, PA, USA). Fluorogenic HDAC1, 2, 3, 4, 6 or 8 assay kits were from BPS Bioscience Corp. (San Diego, CA, USA), colorimetric HDAC activity kits from BioVision Inc. (Milpitas, CA, USA), PGE2 immunoassay kits from R&D Systems (Minneapolis, MN, USA) and IL-6 ELISA kits from eBioscience Inc. (San Diego, CA, USA). The pGL4.32[luc2P/NF-κB-RE/Hygro] and pGL4.30[luc2P/NFAT-RE/Hygro] plasmids were from Promega Corp. (Madison, HI, USA) and the HDAC1-Flag (plasmid 13820) and HDAC6-Flag (plasmid 13823) plasmids from Addgene Inc. (Cambridge, MA, USA). TurboFect transfection reagent was from Fermentas (Burlington, ON, Canada). Unless otherwise stated, all other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
6
NFAT Transcriptional Activity Assay
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RAW264.7 cells were stably transfected with an NFATc1 luciferase reporter construct, pGL4.30 [luc2P/NFAT‐RE/Hygro] (Promega). For luciferase reporter assay, 1.5 × 104 cells/well were seeded into 48‐well plates. After overnight incubation, the cells were serum‐starved for 2 hours and pre‐treated with different concentrations of D.P for 1 hour. Then, the cells were stimulated with 50 ng/mL RANKL for 24 hours. When the time‐point was achieved, the cells were lysed, and luciferase reporter assay was performed using Luciferase Assay System (Promega) as per manufacturer's instructions.
7
STIM1 Luciferase Assay Protocol
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The full-length cDNA of mouse STIM1 was inserted into pEF4/myc-His B (Life Technologies) between BamHI and XbaI sites for luciferase assays. The luciferase reporter pGL4.30[luc2P/NFAT-RE/Hygro] and the pRL-TK plasmid encoding Renilla luciferase were purchased from Promega. The luciferase reporter plasmid was further transfected into Jurkat T cells to make a stable reporter cell line (termed Jurkat-NFAT-Luc).
8
Monitoring Transcription Factor Activation
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Example 4
A DNA fragment encoding NFκB, CREB, or ISRE response sequence synthesized to have a XhoI restriction enzyme recognition sequence on 5′-terminal side and a HindIII restriction enzyme recognition sequence on 3′-terminal side was incorporated into pGL4.26 (available from Promega) harboring firefly luciferase gene, that was cleaved by a treatment with restriction enzymes XhoI and HindIII, to prepare NFκB, CREB and ISRE responsive reporter plasmid. For measuring NFAT activity, a commercially available reporter plasmid (pGL4.30 [luc2P/NFAT-RE/Hygro], available from Promega) harboring firefly luciferase gene incorporated downstream the NFAT response sequence was used.
Stimulation with anti-CD3 antibody or stimulation with anti-CD3 antibody and mSdc1-His was applied to DO11.10-T cells into which the aforementioned reporter plasmid was introduced, and the activity of each of the transcription factor proteins (NFAT, NFκB, CREB and ISRE) after a lapse of 6 hours from stimulation was monitored by change in the activity of a reporter protein luciferase. The result is shown in
As illustrated in
9
Regulation of NFAT Signaling in HEK293 Cells
10
Regulation of NFAT Signaling in HEK293 Cells
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HEK293 cells were cultured in DMEM as described above in 24-well plates. After reaching 40–50% confluence, siRNA oligos were transfected using DharmaFECT (GE Dharmacon) for 48h before further transfection of NFAT-luciferase reporter gene pGL4.30[luc2P/NFAT-RE/Hygro] (Promega). The Renilla luciferase gene (pRL-TK) was cotransfected as a control for counting transfected cells and normalizing the luminescence signals. HEK293 cells were treated with phorbol 12-myristate 13-acetate (PMA; 1 μM) and thapsigargin (1 μM) for 8 hours. Three duplicates were used for each treatment. Cells were then harvested and lysed by following the manufacturer’s protocol. Luciferase activity was assayed using the Dual Luciferase Reporter Assay System (Promega) on a Biotek Synergy2 luminescence microplate reader. The ratio of firefly to Renilla luciferase activity was plotted for HEK293 cells. All the data were normalized against the control group. For the rescue group, after 48 h treatment with siRNA, HEK293 cells were further transfected with the siSTIMATE-resistant variant of mCherry-STIMATE, along with the NFAT-luciferase reporter genes and pRL-TK.
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