The pGL3-CSL construct was kindly provided by Dr. Hyunggee Kim (Korea University, Seoul, Republic of Korea) and contains a CSL binding site. The mouse Notch1 intracellular domain construct, p3xFLAG-CMV7-N1ICD, was kindly provided by Dr. Raphael Kopan (Washington University, St Louis, MO) and contains a N-terminal FLAG epitope tag and amino acids 1744–2531 of the full length Notch1. IL-6 promoter sequence located between −1243 to + 7 8 from the transcription start site was PCR-amplified from genomic DNA purified from NIH 3T3 cells using primers 5’- GGCTCGAGGTGATCCTGAGAGTGTGTTTTG-3’ and 5’-GGAAGCTTAGCGGTTTCTGGAATTGACTATCGTTCTTGG-3’, which includes a XhoI and a HindIII site on the 5’ and the 3’ end, respectively. Promoter fragments were ligated into the XhoI/HindIII site of the pGL4.10 luciferase vector (Promega, Madison, WI) to generate pGL4.10-IL6p. Site-directed mutagenesis was performed using a KOD-Plus-Mutagenesis Kit (TOYOBO) according to the manufacturer’s protocol, to generate an IL-6 promoter reporter construct with mutations in putative CSL binding site located at −116 to −110 (in which 5’-TTTCCCA-3’ was changed to 5’-TTTCCGG−3’). The resultant construct was verified through complete DNA sequencing of both DNA strands. Cells were transduced with empty vector or N1ICD expression vector using Lipofectamine LTX reagent (Invitrogen).
Pgl4.10 luciferase vector
Manufactured by Promega
Sourced in United States
The PGL4.10 luciferase vector is a plasmid designed for the expression of firefly luciferase in mammalian cells. It contains the firefly luciferase gene under the control of the SV40 early promoter, enabling the production of the luciferase enzyme, which catalyzes a bioluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Primestar mutagenesis basal kit, by Takara Bio (2 mentions)
Quickchange 2 kit, by Agilent Technologies (2 mentions)
In fusion cloning kit, by Takara Bio (2 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Victor 3 luminometer, by PerkinElmer (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Prl tk, by Promega (1 mentions)
Pcdna6 v5 hisa, by Thermo Fisher Scientific (1 mentions)
Pcmv tag 2a vector, by Agilent Technologies (1 mentions)
Pmir report luciferase vector, by Promega (1 mentions)
Dual luciferase assay, by Promega (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Endofectin max, by GeneCopoeia (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
16 protocols using pgl4.10 luciferase vector
1
Notch1 Signaling Regulates IL-6 Promoter
2
Smox Promoter Regulation in Hypoxia
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TR-MUL5 cells were seeded in a 96-well plate at 1.5 × 104 cells/well containing 65 µL of 10% FBS/DMEM. After incubation for 24 hours, cells were cotransfected with the X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich) containing the pGL4.10 luciferase vector (Firefly-expressing plasmid; Promega), with the Smox promoter (–1067 to +122 bp from transcriptional start site of Smox), and pRL-CMV vector (Renilla-expressing plasmid; Promega). Cells were incubated under hypoxic or normoxic conditions for 24 hours after waiting 24 hours after transfection. Firefly and Renilla luciferase activities were determined using the Dual-Glo luciferase assay system (Promega), and the luciferase activity (a Firefly: Renilla luciferase ratio) was measured according to the manufacturer's instructions. The promoter sequence analysis revealed six putative hypoxia response elements (HREs), 5′-A/GCGTG-3’, over the Smox promoter region. Subsequently, the Smox promoter reporter with each of the six mutant sites was modified into a pGL4.10 luciferase vector using PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Shiga, Japan). The HRE wild-type or mutated constructs, together with pRL-CMV, were transiently cotransfected into TR-MUL5 cells, followed by treatment with hypoxia, and the luciferase activity was measured.
3
Luciferase Assay for DUSP4 Enhancer Activity
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The DNA sequence underlying the ChIP-Seq-identified DUSP4 EA-SC-binding site was cloned into a pGL4.10 luciferase vector (Promega) behind a DUSP4 promoter. For comparison, a control pGL4.10 vector with the DUSP4 promoter and a piece of genomic DNA of similar size, and located between the DUSP4 TSS and the DUSP4 EA-SC binding site was also generated. These vectors were transfected into HEK293 [ATCC, tested to be mycoplasma free using mycoalert mycoplasma detection kit (Lonza), and used between passages 2–15 after thawing] together with a renilla control vector (pRL-TK, Promega) and increasing amounts of a pCATCH-DC-SCRIPT expression vector. HEK293s were plated 24 h before transfection using metafectene. Cells were harvested after 24 h, and cell lysates were analyzed for luminescence according to the manufacturer’s protocol (Dual Luciferase Reporter assay, Promega) using a Victor3 luminometer (PerkinElmer). Relative light units were calculated after correction for transfection efficiency based on the activity of the cotransfected pRL-TK.
4
Cloning and Expression of TCF4/TCF7L2 and Id2 Enhancer
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The full length, TCF4/TCF7L2 cDNAs were isolated by PCR-based cloning techniques using the following primer sets: TCF4/TCF7L2 (forward, 5′-CTTCCTCCTTCATTTTTCTT-3′; reverse, 5′-CAATGAACTCGATAAACATC-3′). The TCF4/TCF7L2 cDNAs was inserted into pcDNA6/V5-HisA (Invitrogen, Grand Island, NY, USA) for transient expression. The putative Id2 enhancer region was cloned by PCR amplification from the genomic DNA from mouse tail using the forward primer 5′-GCGTTTCCAGGCTGACTTAC-3′ and the reverse primers 5′-AGACCTTGCCAAAGCAAAAG-3′, and inserted into pGL4.10 luciferase vector (Promega, Madison, WI, USA) to generate the reporter construct (Fig. 1 C). The pCMV6-XL5 expression vector carrying the CTNNB1 was obtained from T. Akiyama (The University of Tokyo). Site-directed mutagenesis for mutating the CTNNB1 was performed using PrimeSTAR mutagenesis Basal kit (TaKaRa, Shiga, Japan) with the following oligomers: 5′-CAGTCTTACCTGGACTa TGGAATCCATTCTGGTG-3′ and 5′-CACCAGAATGGATTCCAt AGTCCAGGTAAGACTG-3′. The lower cases indicate the mutated sequences in the CTNNB1 phosphorylation sites. The mutated cDNA were subcloned into pcDNA6/V5-HisA.
5
Quantifying p53 Transcriptional Activity
6
SOHLH2-Induced Sycp1 Promoter Activity
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To measure SOHLH2-induced Sycp1 promoter activity, a 572-bp fragment of Sycp1 promoter ([−470/+ 102] promoter) encompassing three putative E-boxes and transcription start site was PCR-amplified using the primers 5′-CTCGAGAGCACAGGACTCATGTTTGG-3′ and 5′-AAGCTTCGTGACAGAGGTGTGTACGTG-3′. The fragment was first cloned into pTOP TA V2 (Enynomics, Korea) and a 572 bp HindIII-XhoI fragment was subcloned into pGL4.10-luciferase vector (Promega, USA). The DNA fragment encoding SOHLH2 was synthesized using cDNA from mouse testes and cloned to pCMV-Tag 2A vector (Agilent Technologies, USA) to make FLAG-tagged SOHLH2 expression vector. HEK293T cells were seeded into 6-well plates at a density of 6 × 105 cells/well 24 hour prior to transfection. Cells were co-transfected with the indicated amount of SOHLH2 expression vector, 200 ng of Sycp1 promoter luciferase plasmid, and 50 ng of pRL-TK plsmid (Promega) using Lipofectamine 2000 (Invitrogen). After 2 days, cells were harvested, lysed, and analyzed for luciferase activity using dual-luciferase reporter assay system (Promega) and Centro XS3 LB960 microplate luminometer (Berthod Technologies, USA).
7
Cloning of Human αSMA Promoter
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The human αSMA/ACTA2 gene promoter was cloned from human immortalized keratinocyte HaCaT genomic DNA, using hACTA2 promoter −1400/+50 bp-specific primers for PCR: Forward, 5´-AAAAACTCGAGTCAAACAGATCTGA CATAGTAACATGAGTGAACAGCTGGTCATGGC-3´; reverse, 5´- TTTTTAAGCTTCA GGGAAGCTGAAAGCTGAAGGGTTATATAGCCCCTTGG-3´. Amplified DNA was purified by agarose gel electrophoresis and digested by XhoI/HindIII and inserted into the pGL4.10-luciferase vector (Promega Corp., Stockholm, Sweden).
8
Transcriptional Regulation of ENPP2 Promoter
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HEK 293T cells were transfected with vectors encoding for 2.5 kb of ENPP2 promoter. In some constructions κB1 (CTGGCATTTCCAGTAT) or/and κB2 (CTGGTGGAAAGCCCTT) sites were deleted by directed mutagenesis. For analysis of enhancer activity, HEK 293T cells were transfected with the pGL4.10 luciferase vector (Promega, WI, USA) containing a minimal promoter sequence with or without the enhancer region located at chr8:120733232-120735226 on build 37 from ENCODE (Gene synthesis and subcloning, Bio Basic, ON, Canada). In these experiments, a vector coding for renilla luciferase was used as a reporter for transfection efficiency. Luciferase activities were measured at 48 h after transfection with the Promega Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol.
9
Luciferase Reporter Assay for Enhancer Analysis
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The pGL4.10 luciferase vector (Promega, Madison, WI) was used. The enhancer regions were cloned upstream of the luciferase-coding sequence. The reporter constructs were then cotransfected with a control pGL4.74 (Promega) vector expressing Renilla luciferase. The luciferase signal was first normalized to the Renilla luciferase signal and then normalized to the signal of the empty pGL4.10 plasmid. Primers used for cloning were as follows: e6-S, 5′-TGTATGGGTTTCTTCCTGGGCTGT-3′; e6-AS, 5′-CAGTTTTTCAGGTTTCTCTGGGGTCC-3′; e7-S, 5′-TGGGCTTCATGACAGCATCCCTA-3′; e7-AS, 5′-TTGACATGGGCCATTACTCCATCC-3′.
10
Quantifying p53 Transcriptional Activity
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The ~1.2 -kb p21 and ADRB3 genomic fragments containing the p53 binding site were cloned into the pGL4.10-luciferase vector (Promega) using the In-Fusion Cloning kit (Takara). Mutations were introduced into these constructs using the QuickChange II kit (Agilent technologies) (see Table S2 for primer sequences). Luciferase activity was measured in p53−/− 3T3 L1 cells 24 h after cotransfection with wild-type p53, p53 R178C, or empty vector pcDNA and pGL4.74 plasmid containing the TK promoter and Renilla luciferase as a transfection efficiency control (Dual -Luciferase Reporter Assay System, Promega).
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