The fragments of TP53 promoter were amplified and cloned into pGL3.0-Basic Vector (Promega, Madison, WI, USA) for the luciferase reporter plasmids. The fragments were verified using Sanger sequencing. HOXA5-modified cells were seed into 24-well-plate (8 × 105) and co-transfected with above plasmids and pRL-TK plasmids (Promega) using Lipofectamine 2000 (Invitrogen). Cells were collected 48 h post-transfection, and luciferase activities were detected by luminometer (Promega) using the Dual-Luciferase Assay Kit (Promega, Madison, WI). Data were analyzed as relative luciferase activity (Firefly luciferase activity/Renilla luciferase activity). Each experiment was performed in triplicate.
Prl tk plasmid
Manufactured by Promega
Sourced in United States, China, Germany
The PRL-TK plasmid is a molecular biology tool used for gene expression analysis. It contains a promoter sequence from the prolactin (PRL) gene and the thymidine kinase (TK) reporter gene. This plasmid can be used to study the regulation of the PRL gene promoter in various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (121 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (45 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (19 mentions)
Dual luciferase assay system, by Promega (15 mentions)
Pgl3 control vector, by Promega (12 mentions)
Pgl3 basic vector, by Promega (11 mentions)
Lysis buffer, by Promega (10 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (9 mentions)
Dual luciferase reporter assay, by Promega (8 mentions)
Dual luciferase assay kit, by Promega (7 mentions)
Lipofectamine 2000, by Promega (6 mentions)
Pgl3 vector, by Promega (6 mentions)
Passive lysis buffer (plb), by Promega (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dual glo luciferase assay system, by Promega (5 mentions)
Dual luciferase reporter assay kit, by Promega (5 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (4 mentions)
Pgl3 basic luciferase vector, by Promega (3 mentions)
273 protocols using prl tk plasmid
1
TP53 Promoter Transcriptional Activity
2
Constructing Luciferase Reporter Plasmids
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For the construction of luciferase reporter plasmids, we amplified the promoter of NAF1 gene from genomic DNA of SF295 cells and digested the amplification product with restriction enzymes. Then, the digested production was inserted into predigested pGL3-Basic luciferase vector (Promega Corp., WI) to construct the luciferase reporter plasmids pGL3-NAF1-Luc. Supplementary Table S7 presents the primers used for plasmid construct, and the construct was confirmed by Sanger sequencing. To test promoter activity of NAF1 gene modulated by c-Myc, NRF2 and TERT, SF295 cells expressing c-Myc, NRF2 and TERT, and control cells were cotransfected with pGL3-NAF1-Luc or pGL3-Basic-Luc, and pRL-TK plasmids (Promega Corp., WI, USA), which express Renilla luciferase and was used as an internal control to normalize transfection efficiency. Next, we used the dual-luciferase reporter assay system (Promega Corp., WI) to analyze the luciferase activity in accordance with the manufacturer’s instructions. The data were presented as relative luciferase activity (Firefly luciferase activity/Renilla luciferase activity). Each assay was run in triplicate.
3
PAK1 Signaling Pathway Modulation
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The GFP or human GFP‐PAK1 expressing plasmids were kind gifts of Prof. Parsons (Kings College London, UK) and described in Parsons et al.35 PDsRed2‐humanPAK1 was kindly donated by Prof. Jonathan Chernoff (Fox‐Chase Cancer Center, Philadelphia, Pennsylvania, United States) and described in Joseph et al.,32 while the empty vector was purchased from Clontech Laboratories Inc. 4XStat3‐Firefly Luciferase (FLuc) plasmids were kindly donated by Prof. Besser (Max‐Delbrück‐Center, Berlin, Germany). PRL‐TK plasmids were purchased from Promega. C2C12 cells were treated with 1 or 10 μM dexamethasone (Sigma‐Aldrich D2915), as indicated in the legend, or mouse interleukin‐6 (IL‐6) at 10 and 100 ng/mL (SPACE Import Export Srl, 406‐ML‐025) dissolved in water or water for controls. Cells were treated with 10 μM IPA‐3 (Sigma‐Aldrich I2285) dissolved in dimethyl sulfoxide (DMSO) or DMSO for controls. The medium, supplemented with IPA‐3 or DMSO, was refreshed every day or every 2 days depending on the experiment. For experiments shown in Figure 2 , except for Figure 2 B and 2 C, treatments lasted 24 h and started 24 h after cell transfection with PAK1‐expressing plasmids.
4
Evaluating miR-760 Regulation of Cyclin D1
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293T cells were seeded in a 24-well plate and co-transfected with 0.5 μg pmirGLO vector, 80 nM of miR-760-mimic and 1 μl of Lipo3000 (Invitrogen) in 50 μl of Opti-MEM Reduced-Serum Medium (Invitrogen). NC mimic was used as the control. To verify the activation of the cyclin D1 promoter, TRE-containing sequence (5′-AAAATGAGTCAGAA-3′) was cloned into pGL4.27 vector to produce the plasmid pGL4.27-Cyclin D1-wild-type (WT). miR-760 overexpressed in SW620 or HCT116 cells, and control psico-transducted cells were seeded separately in 24-well plates and co-transfected with 0.25 μg pGL4.27-cyclinD1-WT plasmids, 0.25 μg pRL-TK plasmids (Promega), and 1 μl Lipo3000 (Invitrogen) in 50 μl Opti-MEM Reduced-Serum Medium following the manufacturer’s instructions. Twenty-four hours following transfection, the activities of Firefly and Renilla luciferases in cell lysates were measured using the Dual-Glo®Luciferase Assay System (Promega) and the Fluoroskan Ascent FL (Thermo Fischer Scientific). Firefly luciferase activity was normalized to Renilla luciferase activity. All transfection experiments were conducted in triplicate.
5
Transcriptional Regulation Profiling in HepG2 Cells
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HepG2 cells were plated onto 24-well plates at a density of 4 × 104 cells per well. The cells were co-transfected with reporter gene plasmids (pGL3-P0, pGL3-P1, pGL3-P2, pGL3-P3, pGL3-P4, p65-mut 1, p65-mut 2, p65-mut 3, p65-mut 1 + 3, pGL3-P4-UTR-wt and pGL3-P4-UTR-mut) at a dose of 100 ng/well and pRL-TK plasmids (40 ng/well) (Promega) and corresponding siRNAs, miRNA and miRNA inhibitors. The cells were collected after 48 h and the luciferase activity was valuated according to the manufacturer's instructions provided by Promega. Each experiment was repeated at least three times.
6
Bioinformatic Analysis of lncRNA-miRNA-mRNA Interactions
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The starBase v2.0 (http://starbase.sysu.edu.cn/ ) was used in the bioinformatics analysis to predict the binding sites between GATA3-AS1 and miR-30b-5p, and between miR-30b-5p and Tex10 (15 (link)). The sequences of GATA3-AS1 wild-type (wt, chr10:8,092,655-8,092,876) and Tex10 wt (chr9:103,064,259-103,064,483), as well as their corresponding mutant sequences were synthesized and inserted into the pGL3 vectors by Shanghai Sangon Biotech Co., Ltd., and named as pGL3-GATA3-AS1 wt, pGL3-GATA3-AS1 mut, pGL3-Tex10 wt, and pGL3-Tex10 mut plasmids, respectively. Cells (4×104) were co-transfected with indicated pGL3 plasmids and miR-30b-5p mimic or mimic NC, along with pRL-TK plasmids (Promega Corporation), using Lipofectamine 2000 according to the manufacturer's instructions. The luciferase activity was monitored 48 h post transfection using Dual-Glo Luciferase assay (Promega Corporation) according to the manufacturer's instructions. The relative firefly luciferase activity was normalized to Renilla luciferase activity.
7
Dual-Luciferase Assay for WNT1 3'UTR
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The DNA sequences of 3ʹ-UTR-wide type of WNT1 (WNT1-WT) and the corresponding mutant vector 3ʹ-UTR-mutant type of WNT1 (WNT1-MUT) were separately synthesized and cloned into the pGL-3 basic vector (Promega, Madison, WI, USA). Cells were planted into 24-well plates, and then cells were co-transfected with WNT1-WT or WNT1-MUT plasmids, miR-22-3p mimic or miR-NC (RiboBio, Guangzhou, China), and pRL-TK plasmids (Promega, Madison, WI, USA). Cells were collected and then luciferase activity were measured using the Dual-Luciferase Reportor Assay System (Promega, Madison, WI, USA).
8
Regulation of CYP2S1 and CYP1B1 by AhR
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pGL3-CYP2S1-Luc and pGL3-CYP1B1-Luc were created by cloning promoter regions of CYP2S1 and CYP1B1 genes into pGL3-Basic luciferase vector (Promega Corp., WI, USA), and the constructs were then verified by Sanger sequencing. To determine regulatory role of CYP2S1 promoter by AHR, 293T, or 8505C cells expressing AHR or control cells were co-transfected with luciferase reporter plasmids (pGL3-Basic or pGL3-CYP2S1-Luc) and pRL-TK plasmids (Promega Corp., WI, USA). To further determine the effect of CYP2S1 depletion on AHR-mediated transcriptional activation of CYP1B1, BCPAP, and 8505C cells were firstly transfected with siRNAs targeting CYP2S1 or control siRNA, and then co-transfected with luciferase reporter plasmids (pGL3-Basic or pGL3-CYP1B1-Luc) and pRL-TK plasmids. Cell were collected 48 h post-transfection, and luciferase activities were measured using the dual-luciferase reporter assay system (Promega Corp., WI, USA). Primer sequences for dual-luciferase reporter plasmids were presented in Supplementary Table 5 .
9
NF-κB Pathway Activation in SSc Fibroblasts
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Subconfluent fibroblasts in culture were co-transfected with A20-luc, NF-κB mutated A20-luc, and Renilla luciferase pRL-TK plasmids (Promega) using Lipofectamine reagent (Thermo Fisher Scientific)17 (link). Cultures were harvested following indicated incubation period, and whole-cell lysates normalized with pRL-TK were assayed for their luciferase activities The experiment was performed in triplicate and repeated twice with consistent results. In separate experiments, near-confluent SSc fibroblasts were transiently transfected with antisense-DREAM plasmid or control plasmid. Cells were fixed and immunolabelled.
10
Luciferase Assay for Lung Cancer Cell Lines
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The luciferase assays were executed as described previously [26 (link), 30 (link)]. In brief, two human lung cancer cell lines, A549 and NCI-520 were cultured into 24-well plates and then transfected with 1.5μg reporter plasmids (T or C allele) and 10 ng pRL-TK plasmids (Promega, Madison,WI) using lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA). The activity of each reporter with firefly luciferase and the internal standard with Renilla luciferase was quantified with a Dual-Luciferase Reporter Assay System (Promega).
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