The luciferase reporter vector incorporating fragments of the MYC promoter into the pGL4 vector (Promega) was utilized [16 (link)]. The CRISPR activation system (plenti-dCas9-VP64, plenti-MS2-p65-HSF1, and pE1-U6-gRNA-MS2 [28 (link)] of the individual candidate genes) or pT3.5 overexpression vectors were transfected to HEK293T cells expressing the luciferase reporter vector pRL Renilla using the Lipofectamine 3000 reagent (Invitrogen). Cells were collected 48 h after transduction. Luciferase activity was measured by following the protocol of the dual-luciferase reporter assay system (Promega) and Lumat LB9507 (Perkin Elmer). The luciferase activity values were standardized with the luciferase activity value of pE1-h-HPRT.
Pgl4 vector
Manufactured by Promega
Sourced in United States
The PGL4 vector is a plasmid vector designed for use in cell-based reporter assays. It contains the firefly luciferase reporter gene, which can be used to monitor transcriptional activity in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (14 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Dual luciferase assay system, by Promega (4 mentions)
Dual luciferase reporter dlr assay system, by Promega (3 mentions)
Lumat lb9507, by PerkinElmer (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Myc grp78, by Addgene (2 mentions)
Myc perk, by Addgene (2 mentions)
Myc chop, by Addgene (2 mentions)
P stat3 luc, by Beyotime (2 mentions)
Dual luciferase reporter assay, by Promega (2 mentions)
293t cell, by American Type Culture Collection (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Foetal bovine serum (fbs), by Wisent (1 mentions)
Tan 1, by Merck Group (1 mentions)
Dual luciferase reporter assay system e1910, by Promega (1 mentions)
65 protocols using pgl4 vector
1
Luciferase Assay for MYC Promoter Regulation
2
Osteosarcoma Cell Lines and Tan I Assay
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The human OS cell lines U2OS and MOS‐J, and the mouse OS cell line OS‐1 were obtained from ATCC. Stable U2OS‐luc and OS‐1‐luc cell lines were established by transfection with the pGL4 vector (Promega) and selection with G418 (Sigma). The U2OS and MOS‐J cells were cultured in Dulbecco's Modified Eagle Medium (Gibco), while OS‐1 cells were maintained in RPMI‐1640 medium (Gibco). The mediums were supplemented with 10% foetal bovine serum (Wisent); then, the cells were incubated in a humidified incubator with 5% CO2 at 37℃.
Mice were obtained from the Shanghai branch of China's National Rodent Laboratory Animal Resources. All animal experimental protocols were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences, Central South University.
Tan I was purchased from Sigma, and a 50 mmol/L stock solution was prepared in dimethyl sulfoxide (DMSO; Sigma) and stored at −20°C. Different concentrations of Tan I were prepared by defined dilutions in culture medium. Most appropriate antibodies were purchased from Cell Signaling Technology, unless otherwise stated. Antibodies were used according to the manufacturer's instructions.
Mice were obtained from the Shanghai branch of China's National Rodent Laboratory Animal Resources. All animal experimental protocols were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences, Central South University.
Tan I was purchased from Sigma, and a 50 mmol/L stock solution was prepared in dimethyl sulfoxide (DMSO; Sigma) and stored at −20°C. Different concentrations of Tan I were prepared by defined dilutions in culture medium. Most appropriate antibodies were purchased from Cell Signaling Technology, unless otherwise stated. Antibodies were used according to the manufacturer's instructions.
3
SLUG Promoter Luciferase Assay
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The previously reported promoter region for SLUG (−516/+116) was amplified and cloned into the pGL4 vector (Promega, Madison, WI, USA). 293T cells were transfected with the pGL4-Snail/promoter together with pQCXIP-GFP-ALX4 or pQCXIP-GFP-ALXHOXB13 and pRTK-Luc to normalize the transfection efficiency. Forty-eight hours later, the activities of Firefly luciferase and Renilla luciferase were measured using the Dual Luciferase Reporter Assay System (Promega). Luciferase activity was measured in triplicate, and three independent experiments were performed.
4
Cloning USP5 Regulatory Regions
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Genomic DNA was extracted from HCT116 cells according to the manufacturer's protocol (Qiagen). The regulatory sequences of USP5 were predicted by the UCSC Genome Browser website, and different truncated USP5 regulatory regions were amplified by PCR. The primers used for PCR amplification of truncated USP5 regulatory regions were as shown in supplementary Table S2 , and the bold sequence indicated the protection bases and restriction enzyme cutting sites. These fragments were then inserted into the pGL4 vector (Promega, Madison, WI) as described previously 16 (link).
5
Plasmid Construction for ER Stress Signaling
6
VEGF mRNA Regulation by IMP3
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VEGF 5’‐UTR, CDS sequence and 3’‐UTR were amplified and inserted into a pGL4 vector (Promega, USA), and named pGL4‐5’‐UTR, pGL4‐CDS and pGL4‐3’‐UTR, respectively. 293T cells were seeded onto 24‐well plates 24 h before the experiment. Empty vector, pGL4‐3’‐UTR, pGL4‐CDS or pGL4‐5’‐UTR together with AAV2‐IMP3 or AAV2‐Control and Renilla luciferase plasmid were co‐transfected into 293T cells by Lipofectamine 3000 reagent (Invitrogen, USA). Forty‐eight hours post‐transfection, the cells were harvested and lysed. Firefly luciferase activities were detected with a Dual‐Luciferase Reporter Assay System E1910 (Promega, USA) and normalized to control Renilla luciferase levels.
7
SOCS1 Promoter Characterization in Buffalo
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The different fragments of the SOCS1 promoter (GenBank no. NC_059180) derived from primers that hybridize at positions −1999, −1734, −1364, −961, −614 and −77, coupled with a common downstream primer at +105 with Xho Ι and Hind III sites (Table S3 ), were prepared by PCR from mixed genomic DNA, which was isolated from blood samples of 4 buffalo. They were subcloned into pGL4 vector (Promega, Madison, WI, USA) with Xho Ι/Hind III restriction enzyme to generate multiple luciferase reporter vectors (pGL-(−1999/+105), pGL-(−1734/+105), pGL-(−1364/+105), pGL-(−961/+105), pGL-(−614/+105) and pGL-(−77/+105)). The putative transcription factor binding sites were analyzed using the JASPAR database (http://jaspar.genereg.net/ , accessed on 25 April 2022), AliBaba2.1 (http://gene-regulation.com/pub/programs/alibaba2/ , accessed on 25 April 2022) and hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget#!/prediction , accessed on 25 April 2022).
8
Characterization of STAT3-dependent Transcription
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The human transcription factor STAT3 cDNA was cloned into pcDNA3.1 vector with a Myc tag generously as described previously [8 (link)]. The regulatory sequences of cyclin D2 promoter with or without a STAT3 response element was inserted into pGL4 vector (Promega Corporation, Madison, WI, USA) as previously described [34 (link)]. A STAT3-driving luciferase construct (pSTAT3-luc) and a NF-κB luciferase construct (pNF-κB-Luc) were purchased from Beyotime Biotechnology Institute (Nantong, China) [8 (link)]. Plasmids were transiently transfected into HeLa or RPMI-8226 cells by Lipofectamine® 2000 (Invitrogen) according to the manufacturer's instruction.
9
Validation of miR-509-3p Binding to SUMO-3 3'-UTR
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The SUMO-3 3′-UTR fragments with wild-type miR-509-3p binding sites (Wt) or mutated binding sites (Mut) were inserted into a pGL4 vector (Promega). The SUMO-3 3′-UTR PCR product was cloned into the SacI/EcoRV site of the pGL4 vector. The following primers were used to target the SUMO-3 3′-UTR: forward, 5′-TTCACCACGATGATTTTCCT-3′ and reverse, 5′-GCACACAAAAGTACCCACAATATC-3′. The resultant construct was confirmed using DNA sequencing. Site-directed mutagenesis was performed to generate SUMO-3 3′-UTR constructs containing miR-509-3p mutant-binding sites by using the following complementary oligonucleotides: forward, 5′-CTGTAACTTAAATTGGGTTAATCAG-3′ and reverse, 5′-CTGATTAACCCAAT TTAAGTTACAG-3′. A2780CP70 or OVCAR-8 cells were transfected with the vector and the miR-509-3p mimics in combination. We performed luciferase assays 48 h post-transfection using a dual-luciferase reporter assay system (Promega). The normalized luciferase activity was reported as the ratio of luciferase activity to β-galactosidase activity. The activities of firefly luciferase and Renilla luciferase were measured as described previously [12 (link)].
10
Generating c-Maf Mutants and MARE Constructs
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c-Maf was cloned from myeloma cell line LP1, the MafB and USP5 plasmids were purchased from Open Biosystems (Thermo Fisher, Waltham, MA, USA), whereas MafA was cloned from HeLa cells as described previously.11 (link) To generate c-Maf mutants, all lysine (K) residues in c-Maf were mutated to Arginine (R), which generated K0.8 (link) To obtain c-Maf mutants with single lysine residues, the corresponding R was recovered to K by site-directed mutagenesis as described previously.8 (link) K308 c-Maf contained a single K residue, which was present at the 308th amino-acid site. All genes including ubiquitin were subcloned into a pcDNA3.1 vector carrying an HA, a Flag, or a Myc tag. Primers for the specific domains to generate USP5 truncates were designed as shown in Table 1 . The Maf recognition element (MARE, 5′-TGCGAGTGAGGCA-3′) and its mutant version (mtMARE, 5′-gta GAGTg AGtac -3′) were obtained from a previous report.4 (link) A nucleotide sequence containing six tandem MARE or mtMARE was chemically synthesized by Suzhou GeneWiz (Suzhou, China) and it was cloned into a pGL4 vector (Promega, Madison, WI, USA).
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