The luciferase reporter plasmid, 3xERE-TATA-luc, was purchased from Addgene (Cambridge, MA, USA).27 (link) T47D-pMIG or T47D-ING4 cells were co-transfected with the linearized luciferase reporter plasmid and a neomycin resistance gene containing plasmid, pLNCx (Clontech, Mountain View, CA, USA) using Effectene (Qiagen Valencia, CA, USA) and were selected in the media containing 400 mg/mL Geneticin (Gibco, Billings, MT, USA). Cells plated at 50% confluency in a 24-well dish were hormone-deprived in the media containing 10% of charcoal-stripped FBS for 48 h and with or without 100 nM E2 for additional 24 h. Luciferase activity in 30 μL of cell lysates was measured using a Steady-Glo Luciferase Assay System (Promega Corporation, Fitchburg, WI, USA) and Victor3 luminometer (Perkin Elmer Life Sciences Products, Boston, MA, USA). Total protein concentration in cell lysates was measured using the BCA Protein Assay Kit (Pierce Thermo Fisher Scientific, Waltham, MA, USA). Luciferase activity was calculated as relative light units per microgram of protein and normalized to the luc2 gene copy number integrated into the genome as described previously.25 (link)
Ere tata luc
Manufactured by Addgene
Sourced in United States
The 3X ERE TATA luc is a plasmid that contains a luciferase reporter gene under the control of a minimal TATA promoter and three copies of the estrogen response element (ERE). This plasmid is commonly used to study estrogen receptor-mediated gene expression in cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
Plncx, by Takara Bio (1 mentions)
Effectene, by Qiagen (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Transit 2020 reagent, by Mirus Bio (1 mentions)
Infinite f200, by Tecan (1 mentions)
Prl tk plasmid, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Bright glo luciferase substrate, by Promega (1 mentions)
Cell titer glo luminescent viability assay kit, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Phrl tk, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
7 protocols using ere tata luc
1
Luciferase Assay for Estrogen Receptor Transcriptional Activity
2
Transient Transfection of ER-Responsive Luciferase
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For transient transfection, we used pGL2.TATA.Inr.luc plasmid, which contains three copies of vitellogenin oestrogen response element (ERE) upstream of the TATA promoter (3X ERE TATA luc; Addgene plasmid 11354). This is the same plasmid used to construct pGL2.TATA.Inr.luc.neo, used to create the stable cell line T47D-KBluc.
TamR3 and TamR6 cells were grown in phenol red–free RPMI 1640 supplemented with 10% CSS and 1 μM tamoxifen. The cells were seeded on a 96-well plate (2 × 104 cells/well). After 24 hours, the cells were cotransfected with 50 ng each of the ERα-responsive luciferase plasmid and a constitutive Renilla reporter (to normalise for variations in transfection efficiency) using TransIT-2020 reagent (Mirus Bio, Madison, WI, USA). Cells were treated the next day with the test compounds in the presence of 1 nM E2. The compounds were added in a twofold dilution ranging from 0.1 to 50 μM. Tamoxifen was added at concentrations ranging from 0.000095 to 6 μM, and fulvestrant was added in the range of 0.000095 to 50 μM. The medium contained 0.1% (v/v) ethanol and 0.1% (v/v) DMSO. Twenty-four hours after treatment, the medium was aspirated and the cells were lysed by adding 50 μl of 1× passive lysis buffer (Promega). Luciferase activities were assayed using the Dual-Luciferase Reporter Assay System (Promega).
TamR3 and TamR6 cells were grown in phenol red–free RPMI 1640 supplemented with 10% CSS and 1 μM tamoxifen. The cells were seeded on a 96-well plate (2 × 104 cells/well). After 24 hours, the cells were cotransfected with 50 ng each of the ERα-responsive luciferase plasmid and a constitutive Renilla reporter (to normalise for variations in transfection efficiency) using TransIT-2020 reagent (Mirus Bio, Madison, WI, USA). Cells were treated the next day with the test compounds in the presence of 1 nM E2. The compounds were added in a twofold dilution ranging from 0.1 to 50 μM. Tamoxifen was added at concentrations ranging from 0.000095 to 6 μM, and fulvestrant was added in the range of 0.000095 to 50 μM. The medium contained 0.1% (v/v) ethanol and 0.1% (v/v) DMSO. Twenty-four hours after treatment, the medium was aspirated and the cells were lysed by adding 50 μl of 1× passive lysis buffer (Promega). Luciferase activities were assayed using the Dual-Luciferase Reporter Assay System (Promega).
3
Lentiviral Vector Production and Titration
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The CMV promoter downstream of eGFP was substituted from the pLKO-eGFP vector by an ERE promoter (3X ERE TATA luc from AddGene, USA) to drive eGFP expression. For lentiviral vector production 80% confluent 293T cells were transfected for 4 h with 40 μg of a plasmid encoding the viral backbone (pHR’SIN), 10 μg of VSV-G envelope glycoprotein (pMDG.2) and 30 μg of packaging proteins (gag-pol, rev, tat, in pCMVΔR8.74) using 1 μM polyethyleneimine as a transfection agent. Cells were washed and refreshed with Dulbecco’s Modified Eagle Medium-DMEM containing glutamine, 10% Fetal Calf Serum and antibiotic mix. Viral particles were harvested after 36 h, filtered and stored frozen in 100 μl aliquots. For titration, ten-fold serial dilutions of the viral ERE-GFP construct (from undiluted to a dilution of 10−7) were done in DMEM Seed 6*104 293T Cells in each well of the 24-well cluster plate. After 72h cells were fixed with 4% paraformaldehyde for 30 minutes and immunostained for GFP.
4
Transcriptional Activity Measurement
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AR transcriptional activity was determined by ARE-luciferase (ARR3tk-eGFP/SV40-mCherry; 132360; Addgene, Watertown, MA, USA) [23 (link)]. ER transcriptional activity was determined by ERE-luciferase (3X ERE TATA luc; 11354; Addgene, Watertown, MA, USA) [24 (link)]. The pCMV-Green Renilla Luc vector (pCMV-Ren; 16153; Thermo Fisher Scientific, Waltham, MA, USA) was used for normalization. The ratio of ARR3tk-eGFP/SV40-mCherry to pCMV-Ren and 3X ERE TATA luc to pCMV-Ren was 100:1. Dual-Luciferase® Reporter Assay System (E1910; Promega, Madison, WI, USA) was used, and the signal was captured and recorded by microplate reader Infinite F200 (Tecan, Seestrasse, Switzerland).
5
Estrogen Receptor Transcriptional Regulation
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MCF-7 cells were seeded onto 24-well plates at a density of 2 × 105 cells/well and cotransfected with 1 μg of the 3× ERE TATA Luc (oestrogen-responsive element-containing luciferase reporter) construct (Addgene, http://www.addgene.org , plasmid 11354) [27 (link)] and 300 ng of the control pRL-TK plasmid (Promega Italia S.r.l., Milano, Italy) for normalization of transfection efficiency. The putative promoter region of the human FGFR2 gene (−1103 to +459 relative to the transcriptional initiation site) was amplified and inserted into a luciferase reporter vector, as previously described [26 (link)]. Transfections with ERE-luc or pKGFR construct were carried out in triplicate using Lipofectamine 2000 (Invitrogen) following manufacturer's instructions. Luciferase activities were determined with Dual Luciferase Reporter Assay System (Promega) 24 hrs after treatment, according to manufacturer's protocol.
6
Estrogen Receptor Activity Assay in MCF-7 Cells
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Luciferase reporter assay was conducted as previously described [18 (link)]. Briefly, MCF-7 cells were seeded at 15,000 cells/well in 96-well plates in phenol red-free medium supplemented with 5% charcoal-stripped FBS (csFBS) (Gibco, A3382101). After 24 h, the cells were transfected with 3× ERE TATA Luc (#11354; Addgene) using Lipofectamine 3000 (L3000001; Invitrogen). The transfected cells were incubated for 5 h and then treated with EAs at 1 μM final concentration. After 24 h of incubation, 50 μl of Promega Bright-Glo luciferase substrate dissolved in lysis buffer (Promega, E2610) was added to the cells. For the cell viability assay, MCF-7 cells were seeded at 15,000 cells/well in 96-well plates in a phenol red-free medium supplemented with 5% csFBS and 10 nM E2. After 24 h, the cells were treated with 1 μM EAs. Cell viability was determined using the CellTiter-Glo Luminescent Viability Assay kit (Promega, G7570) after 48 h of incubation. The luminescence was measured using a CLARIOStar Plus microplate reader.
7
Toxoplasma Modulation of Estrogen Signaling
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MEFs or MCF7 cells were transiently transfected with 3× ERE TATA luc (Addgene plasmid no. 11354) and phRL-TK (Promega) for 24 h. Cells were then either mock infected or infected with Toxoplasma (MOI of 4) and treated with the vehicle or estrogen (100 nM) and/or tamoxifen (5 µM). After 24 h, ER element activity was assessed with the Dual-Glo luciferase assay system (Promega).
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