To investigate the regulating function of miR-488 on FSCN1, cells were co-transfected with pGLC-FSCN1-3′UTR or pGLC-FSCN1-3′UTR-Mut, miR-488 mimic or inhibitor, and pRL-SV40 (Promega, USA), using Lipofectamine 3000 (Invitrogen, USA). And to explore the transcriptional regulation of miR-488 expression, cells were co-transfected with pGLE-miR-488-pro or pGLE-miR-488-pro-Mut, pCLE-N3ICD or pCLE (vector), and pRL-SV40 (Promega, USA), using Lipofectamine 3000 (Invitrogen, USA). Luciferase and Renilla signals were examined 36 h after transfection using a Dual-Luciferase Reporter Assay Kit (E1910, Promega) according to the manufacturer’s protocol.
Prl sv40
Manufactured by Promega
Sourced in United States, China, Germany, France
The PRL-SV40 is a plasmid that contains the SV40 large T antigen gene, which is commonly used for the immortalization of various cell lines. The PRL-SV40 plasmid can be used to establish stable cell lines that maintain the expression of the SV40 large T antigen.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (143 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (63 mentions)
Dual glo luciferase assay system, by Promega (24 mentions)
Dual luciferase assay kit, by Promega (21 mentions)
Dual luciferase assay system, by Promega (18 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (17 mentions)
Pgl3 basic, by Promega (17 mentions)
Dual luciferase reporter assay, by Promega (12 mentions)
Dual luciferase reporter assay kit, by Promega (10 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (10 mentions)
Passive lysis buffer (plb), by Promega (10 mentions)
Dual glo luciferase reporter assay system, by Promega (9 mentions)
Glomax 20 20 luminometer, by Promega (8 mentions)
Luciferase assay system, by Promega (7 mentions)
Prl tk, by Promega (7 mentions)
Prl cmv, by Promega (7 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (7 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (6 mentions)
Opti mem, by Thermo Fisher Scientific (6 mentions)
Pgl3 basic vector, by Promega (6 mentions)
378 protocols using prl sv40
1
Regulation of FSCN1 by miR-488
2
Quantifying Wnt/β-catenin Transcriptional Activity
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The transcriptional activity of Wnt/β-catenin signaling was assessed using TOPflash and FOPflash reporters (Upstate Biotechnology, Lake Placid, NY, USA). TOPflash includes six wildtype β-catenin/TCF-binding sites upstream of a luciferase reporter, while FOPflash containing six mutant β-catenin/TCF-binding sites is employed as the control of TOPflash [25 (link)]. When cells reached 70–90% confluency in 24-well plates, pcDNA3.1-DVL3 or shDVL3 was co-transfected with TOPflash plus pRL-SV40 or FOPflash plus pRL-SV40 into CRC cells for 48 h. Subsequently, we used to a dual-luciferase assay (Promega Corporation, Madison, WI, USA) to test the activities of TOPflash and FOPflash reporters. The reporter activity of each sample was normalized against Renilla reporter pRL-SV40 (Promega Corporation, Madison, WI, USA) activity to monitor transfection efficiency.
3
Regulation of MAFG-AS1 by miR-339-5p
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A firefly luciferase reporter plasmid (pmirGLO-MAFG-AS1-WT or pmirGLO- MAFG-AS1-MUT) and a renilla luciferase vector (pRL-SV40, Promega) plus small RNAs (miR-339-5p mimics or negative control RNAs) were co-transfected into T47D or MCF-7 cells with Lipofectamine®2000 (Invitrogen, CA, USA). Three independent transfection experiments were performed, and each was done in triplicate. Firefly luciferase activities derived from pmirGLO-control-derived plasmids were normalized to renilla luciferase activity from pRL-SV40 using a luciferase assay system (Promega, WI, USA).
4
PPAR-γ Transactivation Assay in HEK293T
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The HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% v/v penicillin and streptomycin. The cells were grown to 80% confluency at 37 °C, under an atmosphere containing 5% CO2 and then were plated in 96-well plates (15,000 cells per well). For luciferase assays, the HEK293T cells were transfected with 50 ng pcDNA3-PPARγ WT, 70 ng PPRE-X3-TK-luc (1015; Bruce Spiegelman, Addgene, Cambridge, MA, USA), and 10 ng pRL-SV40 (Promega, Wallisellen, Switzerland) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The cells were incubated for 24 h and then treated with 10 μM, 1.25 μM, 156.26 nM, 19.53 nM, and 2.44 nM butyrolactone I or pioglitazone. The luciferase activity was determined 20 h later with the Dual-Glo® Luciferase Assay System 2000 (Promega), according to the manufacturer’s instruction, and the obtained results were normalized with the Renilla luciferase signal obtained with the pRL-SV40 plasmid. The relative luciferase units of treated cells over DMSO-treated control cells were plotted.
5
Luciferase Assay for p53 Activity
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A luciferase reporter plasmid with p53 binding sites, PG13-luc [42 (link)] was a gift from Bert Vogelstein (Addgene plasmid #16442). A control Renilla luciferase plasmid, pRL-SV40, was purchased from Promega (Madison, WI, USA).
Luciferase reporter assays of transiently transfected cells were performed essentially as described previously [18 (link), 43 (link)]. In brief, cells were electroporated with PG13-luc as well as pRL-SV40 and treated with or without the p53 inhibitors and etoposide as described before harvesting for the luciferase assay using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
Luciferase reporter assays of transiently transfected cells were performed essentially as described previously [18 (link), 43 (link)]. In brief, cells were electroporated with PG13-luc as well as pRL-SV40 and treated with or without the p53 inhibitors and etoposide as described before harvesting for the luciferase assay using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
6
PPARγ Transcriptional Activity Assay
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HEK293T cells were cultured in Dulbecco Modified Eagles’ Medium (DMEM; glucose 1 g/L) supplemented with 10% fetal bovine serum and 1% (v/v) penicillin and streptomycin. Cells were grown to 80% confluency at 37 °C under an atmosphere containing 5% CO2 and then plated in 96-well plates (10,000 cells per well). For luciferase assays, HEK293T cells were transfected with 50 ng pcDNA3-PPARγ WT or mutants (R280C, C285Y, Q286E, Q286P, F287Y, R288C, R288H and S289C), 70 ng PPRE-X3-TK-luc (1015; Bruce Spiegelman, Addgene, Cambridge, MA, USA) and 10 ng pRL-SV40 (Promega, Wallisellen, Switzerland) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in opti-MEM according to the manufacturer’s instruction. The media were changed to the culture media after 4 h and the cells were incubated for additional 48 h. Luciferase activity was determined using Dual-Glo® Luciferase Assay System 2000 (Promega), according to the manufacturer’s instruction and the resulting signals were normalized with Renilla luciferase signals obtained from pRL-SV40 plasmids. Relative luciferase units were plotted.
7
Hypoxia-responsive element reporter assay
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Twenty-four hours after seeding at 2 × 105 cells/well in 6-well plates, cells were transiently transfected using the Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s protocol. The nHRE+ and nHRE− vectors were co-transfected with pRL-SV40 (Promega) as a control vector for transfection efficiency, because in preliminary experiments we found that the expression of pRL-SV40 was less influenced by hypoxia than pRL-TK (Promega) and pRL-CMV (Promega) were. On the other hand, dual-luc-nHRE vectors containing both firefly and Renilla luciferases were transfected on their own. Twelve hours later, the culture dishes were washed in growth medium twice and cells were exposed to hypoxic conditions (1, 2, 4, 8, and 16 % O2 or DFO 20, 40, 80, 160, and 320 µg/mL growth medium). Immediately after hypoxic stress, cell lysates were prepared with 250 µL of passive lysis buffer using the Dual Luciferase Assay kit (Promega), and luciferase activities were measured with a luminometer according to the manufacturer’s protocol. The results were expressed as the relative ratio of firefly luciferase activity to Renilla luciferase activity for correction of transfection efficiency in experiments using nHRE+ and nHRE−, and as the ratio of bidirectional activity in experiments using dual-luc-nHRE. These experiments were performed in triplicate.
8
Luciferase Assay for Genetic Variant Analysis
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For the luciferase assay, 293T and PIG1 cells were seeded in 24‐well plates (1 × 105 cells per well) and transfect with 0.8 μg of the recombinant pGL3 reporter vector with either −653 T or −653 C allele using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA). As an internal control, all plasmids were co‐transfect with 0.02 μg pRL‐SV40 (Promega). The pGL3‐Basic vector without an insert and pGL3‐Control vector (Promega) were used as a negative control and a positive control respectively. Cells were collected 48 h after transfection, and cell lysates were prepared according to the manufacturer's instructions. Luciferase activity was measured with a Dual‐Luciferase Reporter Assay System (Promega) and normalized to the activity of pRL‐SV40 with the Renilla luciferase gene. Independent triplicate experiments were performed for each construct.
9
Luciferase Reporter Assay for BDNF-AS
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OE19 cells and OE33 cells were cotransfected by firefly luciferase reporter plasmid (BDNF‐AS‐WT, BDNF‐AS‐MUT,) and a renilla luciferase vector (pRL‐SV40, Promega) plus negative control and small RNAs (NC, miR‐214) using Lipofectamine 2000 (Invitrogen, USA). Experiments were carried out at least 3 times. The luciferase activities from pGL3‐control‐derived plasmids were standardized to renilla luciferase activity from pRL‐SV40 by the luciferase assay system (Promega, USA). After post‐transfection for 48 hours, luciferase activity was evaluated in the harvested cells using the Multimode Detector reporter assay system (Beckman Coulter, WI, USA).
10
PANC1 Cell Transfection and Luciferase Assay
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