Estradiol‐17β (E2) was purchased from Sigma‐Aldrich (St Quentin, France). 4‐hydroxy‐tamoxifen (OHTam) and ICI 182 780 (ICI) were kindly donated by Sanofi‐Aventis and AstraZeneca, respectively. Zeocin was from InvivoGen (Toulouse, France). The FLAG‐tagged full length HDAC9 (HDAC9FL) plasmid was a kind gift from A. Zelent (Petrie et al., 2003 ). For stable transfections, the pcDNA3.1‐HDAC9‐zeo plasmid was obtained by subcloning HDAC9FL in the pcDNA3.1zeo vector (Invitrogen, Cergy Pontoise, France) that confers Zeocin resistance. The ERα promoter luciferase reporter plasmid was from B. Westley (Donaghue et al., 1999 ). The 17EBLuc+ luciferase reporter plasmid contains one copy of the consensus estrogen‐responsive element (ERE). The pRL‐CMV vector (Promega, Charbonnières, France) was used for normalization.
Prl cmv vector
Manufactured by Promega
Sourced in United States
The PRL-CMV vector is a plasmid designed for protein expression in mammalian cells. It contains the cytomegalovirus (CMV) promoter, which drives the transcription of the gene of interest. The vector also includes a multiple cloning site for the insertion of the target gene and a selection marker for transfected cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (31 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Pgl3 basic vector, by Promega (8 mentions)
Dual luciferase reporter assay kit, by Promega (5 mentions)
Passive lysis buffer (plb), by Promega (4 mentions)
Pgl3 control vector, by Promega (3 mentions)
Dual luciferase assay system, by Promega (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Infinite m1000, by Tecan (2 mentions)
Pgl4 basic vector, by Promega (2 mentions)
Pgl3 luciferase reporter vector, by Promega (2 mentions)
Renilla luciferase, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
17β estradiol e2, by Merck Group (1 mentions)
Pcdna3.1zeo vector, by Thermo Fisher Scientific (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
Zeocin, by InvivoGen (1 mentions)
66 protocols using prl cmv vector
1
Estrogen Receptor Regulation Assay
2
Evaluating miR-502 Regulation of DNA Repair Genes
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Hela cells were transfected with DNA plasmids (pLVX-DsRed, pMIR-REPORT, pRL-CMV) using Rotifect+ (Carl Roth CL21.2) and processed according to the Dual-Luciferase Reporter Assay protocol (Promega E1910). Pre-miR-502 was expressed from pLVX-DsRed-Monomer-C1 vector (Clontech) cloned downstream of the dsRED gene and 3′UTRs of Ku70, Ku80, LIG4 and XLF were fused downstream to the firefly luciferase gene in the pMIR-REPORT vector (Ambion). pRL-CMV vector was cotransfected to account for transfection efficiency and cell death (Promega).
3
Twist1 Promoter Transcriptional Activity
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Cells (1.5 × 104 cells/ well) were cotransfected with 0.4 μg of pcDNA3.1-STS, or TCF/LEF TOPFLASH reporter plasmid vector according to the manufacturer’s protocol using the NeonTM transfection system (Invitrogen). The Twist1 promoter vector was provided by Dr. Mien-Chie Hung (University of Texas, USA). The Twist-HRE mutant promoter vectors were kindly supplied by Dr. H.Y. Lee (Konyang University, Korea). pRL-CMV vector (Promega) was cotransfected as an internal control. After 24 h, the cells were harvested and luciferase activities were measured using the Dual Luciferase Assay System (Promega) with a SynergyTM H1 hybrid microplate reader (Biotek, Winooski, VT).
4
Characterization of CCND2 3'UTR Regulation
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Wild-type (WT) human CCND2 3′-UTR luciferase reporter vector was obtained from OriGene (Rockville, MD). Mutations (MUT) of the miR-206 binding site in the CCND2 3′-UTR sequence were created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The full-length of OTUD6B-AS1 was amplified and cloned into the pGL3-basic vector (Promega, Madison, WI). After cervical cancer cells were seeded onto 24-well plates, 100 ng of the luciferase reporter vector, 10 ng of the pRL-CMV vector (Promega, Madison, WI), as well as 30 nM of miR-206 mimic, control mimic, miR-206 inhibitor, or control inhibitor, were transfected to cancer cells using Lipofectamine 2000 (Invitrogen). At 48 h after transfection, luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega).
5
Mapping miRNA Binding Sites in 3'UTRs
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The 3’UTRs of NlInR1 and NlInR2 were amplified and introduced into the pMIR-REPORT vector (Obio, China) downstream of the firefly luciferase gene. The pRL-CMV vector (Promega, USA), which contains the Renilla luciferase gene, was used as a control luciferase reporter vector. The predicted binding site (5’-CACTAGTGACCGCGTAGTTGCCTGCCG-3’) was completely removed for mutant 1, and another binding site (5’-TGACGTTGCCGCCGCCACTGCCG-3’) was removed for generation of mutant 2. HEK293T cells (Obio, China) were cultured at 37°C in 5% CO2 in DMEM (Gibco, USA) media supplemented with 10% FBS (Hyclone) for 24 h in 96-well culture plates. Cells were transfected with pMIR-REPORT (0.2 μg), pRL-CMV (0.01μg), 0.25 μl of 100 nM miRNA mimics (RiboBio, China), and 0.25 μl Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. The activity of the two luciferase enzymes was measured 48 h after transfection following the manufacturer’s recommendations (Dual-Luciferase Reporter Assay System, Promega, USA) with Infinite M1000 (Tecan, Switzerland). Three replicates were performed for each experiment. Results are expressed as the means of the ratio of firefly luciferase activity/Renilla luciferase activity. Controls were set to one, and the two-tailed t-test was used for statistical analysis.
6
Dual Luciferase Assay for TGF-beta Signaling
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The pGL3-SBE4-Luc reporter plasmid was kindly provided by Dr. Bert Vogelstein (Baltimore, MD). This reporter has 4 copies of the Smad4 binding site of Vestigial (Vg) Drosophila gene palindrome sequences on the promoter of firefly luciferase, cloned in the pBV-Luc vector [38 (link)]. The pGL2-3TP-Luc construct contains a synthetic promoter composed of a TGF-β-responsive fragment of the promoter of the human plasminogen activator inhibitor-1 (PAI-1 or serpine1) gene inserted downstream of three phorbol ester-responsive elements [13 (link)], kindly provided by J. Massague (New York, NY). Dual luciferase firefly/renilla assays were performed with co-transfection of 150 ng of the respective reporter construct and 20 ng of pRL-CMV VECTOR (Promega, Madison, WI). Luciferase activity was normalized to constitutive renilla luciferase activity (pRL-CMV).
7
Investigating miR-646 Regulation of NOB1 3'-UTR
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3′-UTR of NOB1 and a mutation sequence were amplified by PCR using the primers that included a Bgl II restriction site on the 5′ and 3′ strands. The PCR products were inserted into the Bgl II sites of the pGL3-control vector (Promega, Madison, WI, USA) and identified by DNA sequencing. The wild-type plasmid was created containing the 3′-UTR of NOB1 with complementary sequence of miR-646 (pGL3-NOB1 3′-UTR wild), and a mutant plasmid with the mutation sequence without complementary sequence of miR-646 (pGL3-NOB1 3′-UTR mut). Following primers were used: (Forward) NOB1-3′-UTR wild-F, 5′-CAAGCTTAGCGAGTTCCCGCAGGCAAAT-3′ (Reverse) NOB1-3′-UTR wild-R, 5′-CTCTAGACATGATCTCTGGGCACAC-3′ (Forward) NOB1-3′-UTR mut-F, 5′-CAAGCTTAGCGAGTTCCCGCAGGCAAAT-3′ (Reverse) NOB1-3′-UTR mut-R, 5′-CTCTAGACATGATCTCTTTTCACACAGC-3′.
For the luciferase reporter assay, the human malignant renal cell line 786-O was seeded on 24-well plates and co-transfected using Lipofectamine 2000 (Invitrogen) with 100 ng per well of the resulting luciferase UTR-report vector, 2 ng per well of pRLCMV vector (internal control, Promega) and 20 ng per well of miR-646 precursor molecules or control precursor (Applied Biosystems) following the manufacturer's instructions. After 24 h the cells were lysed and the relative luciferase activity was assessed with the Dual-Luciferase Assay Reporter System (Promega).
For the luciferase reporter assay, the human malignant renal cell line 786-O was seeded on 24-well plates and co-transfected using Lipofectamine 2000 (Invitrogen) with 100 ng per well of the resulting luciferase UTR-report vector, 2 ng per well of pRLCMV vector (internal control, Promega) and 20 ng per well of miR-646 precursor molecules or control precursor (Applied Biosystems) following the manufacturer's instructions. After 24 h the cells were lysed and the relative luciferase activity was assessed with the Dual-Luciferase Assay Reporter System (Promega).
8
RNA Synthesis for Virus Replicons
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Sub-genomic reporter replicons: RNA for electroporation was generated from 5 µg of pFK-plasmid (linearized with MluI (NEB)) in a 100 µl reaction mix containing 20 µl of 5× rabbit reticulocyte lysate buffer (400 mM Hepes (pH 7.5), 60 mM MgCl2, 10 mM spermidin, 200 mM DTT), 12.5 µl 25 mM NTP-solution, 2.5 µl RNasin (40 U/µl), 0.1 U pyrophosphatase (Sigma Aldrich), and 6 µl of T7 polymerase. The reactions were performed over night at 37 °C. Subsequently, the input DNA was removed using RQ1 RNase-free Dnase (Promega). The RNA was purified by acid phenol:chloroform extraction and isopropanol precipitation. The pellet was washed with 70% ethanol and resuspended in RNAse free water.
Capped Renilla mRNA for translation experiments: For capped Renilla transcripts, the pRL-CMV vector (Promega) was linearized with BamHI (NEB). The setup of the transcription reaction was as stated above, except 12.5 mM m7G analog (NEB) were added to the reaction, and the GTP concentration in the rNTP stock was reduced to 12.5 mM.
Capped Renilla mRNA for translation experiments: For capped Renilla transcripts, the pRL-CMV vector (Promega) was linearized with BamHI (NEB). The setup of the transcription reaction was as stated above, except 12.5 mM m7G analog (NEB) were added to the reaction, and the GTP concentration in the rNTP stock was reduced to 12.5 mM.
9
Dual-Luciferase Reporter Assay Protocol
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All constructs for the reporter gene assay contained the firefly luciferase gene. For a transient assay, the pRL‐CMV vector (Promega), in which the Renilla luciferase gene was driven by the CMV promoter, was co-transfected with experimental constructs. Luciferase activity was measured 2 days after transfection with a Dual-Luciferase Reporter Assay System (Promega) using Lumat LB9507 (Berthold, Bad Wildbad, Germany). The value of firefly luciferase was normalized to that of Renilla luciferase for adjusting transfection efficiency in each trial. For stable cells, the value of firefly luciferase was normalized to the protein amount that was determined by using a BCA Protein Assay Kit (Thermo Fisher Scientific).
10
Dual Luciferase Assay in HEK293T Cells
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For dual luciferase analysis, ~1 × 105 HEK293T cells were seeded on 24-well plates. Each well was transfected with 400 ng luciferase reporter plasmid and 2 ng of internal control plasmid pRL-CMV vector (Promega, USA) using Lipofectamine 2000. Twenty-four hours later, cells were transfected with pHAGE-puro or pHAGE-puro-ERβ plasmid for another 24 h. Next, cells were harvested and lysed with passive lysis buffer (Promega) and Luciferase units were measured by the dual luciferase assay system protocol (Promega).
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