HEK293FT cells were seeded in 48-well plates at 6.0 × 104 cells/well using steroid-free medium and cultured overnight. For determination of CRE- or androren response element (ARE)-mediated transcriptional activity, cells were transfected with β3-AdR expression vector (pcDNA3.2-β3-AdR-V5 (74 (link))), AR expression vector (pcDNA3.1-AR (68 (link)), pcDNA3.1-AR (K630A/K632A/K633A), pcDNA3.1-AR (C619Y) (32 (link), 75 (link)), pcDNA3.1-AR (L26A/F27A) (76 (link)), pcDNA3.1-AR (E897Q) (76 (link)), pcDNA3.1-AR (ΔpolyQ+Q6+Q5) (35 (link)), pcDNA3.1-AR (C806A)), luciferase reporter vector (p4xCRE-TATA-Luc2P (74 (link)), pGL4-ARE2-TATA-Luc (77 (link)), or pUCP1-pro-Luc2P), and Renilla luciferase reporter vector (pGL4.74[hRluc/TK] (Promega)) with PEI MAX (Polysciences Inc) and Opti-MEM (Thermo Fisher Scientific) for 24 h. For the mammalian two-hybrid assay, cells were transfected with pGAL-CREB, pcDNA3.1-AR or pACT-AR (NTD) (76 (link)), pG5luc (Promega), and pGL4.74[hRluc/TK] with PEI MAX and Opti-MEM for 24 h. Cells were incubated in the presence of 10 nM DHT for 16 h and then stimulated with 10 μM CL316243 or 10 μM forskolin for an additional 4 h. Cells were treated with the AR antagonist bicalutamide (10 μM) 30 min prior to DHT treatment. Luciferase reporter activity was determined as described previously (68 (link)). Data are expressed as relative light units.
Pgl4.74 hrluc tk
Manufactured by Promega
Sourced in United States
The PGL4.74[hRluc/TK] is a laboratory equipment product from Promega. It is a plasmid that contains the Renilla luciferase (hRluc) and Herpes simplex virus thymidine kinase (TK) reporter genes.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (16 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Dual glo luciferase assay system, by Promega (4 mentions)
Opti mem, by Thermo Fisher Scientific (3 mentions)
Pei max, by Polysciences (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Pgl4.32 luc2p nf κb re hygro, by Promega (3 mentions)
Opti mem, by Promega (2 mentions)
Fugene hd, by Promega (2 mentions)
X tremegene hp dna transfection reagent, by Roche (2 mentions)
Optiplate 96, by PerkinElmer (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Pgl4.10 luc2, by Promega (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Pci mammalian expression vector, by Promega (2 mentions)
Pfuultra fusion dna polymerase, by Agilent Technologies (2 mentions)
Pgl4.49 luc2p tcf lef re hygro, by Promega (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Thedual luciferase reporter assay system, by Promega (2 mentions)
Dual luciferase assay system, by Promega (2 mentions)
58 protocols using pgl4.74 hrluc tk
1
Transcriptional Regulation via AR and β3-AdR
2
TEAD Luciferase Assay for YAP/TAZ Activity
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For the TEAD luciferase reporter assay, 1 × 105 DGCs or GSCs were seeded in each well of a 24-well plate. After 12 h, the cells were transfected with the YAP/TAZ-responsive TEAD Firefly luciferase reporter vector 8 × GTIIC-luciferase (Addgene, #34615) (150 ng/cm2) and Renilla luciferase control reporter vector pGL4.74 [hRluc/TK] (Promega) (100 ng/cm2) using TransIT-LT1 (Mirus Bio) in accordance with the manufacturer’s instructions. At 24 h after transfection, Firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay System (Promega) in accordance with the manufacturer’s instructions.
3
Investigating A20 Mutant Effects on Cytokine Secretion
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Expression plasmids encoding C243Y or wild-type (WT) A20 were constructed, as reported previously.22 (link) Monocytic leukaemia THP-1 cells were cultured in RPMI 1640 (Life Technologies, Carlsbad, California, USA), 10% heat inactivated fetal bovine serum (FBS), penicillin and streptomycin. An Amaxa Nucleofector (Amaxa, Cologne, Germany) was used to transfect 1×106 cells with 1 µg of pcDNA3, pcDNA3-A20, or pcDNA3.1-C243Y A20 together with 100 ng of pGL4.74[hRluc/TK] (Promega, Madison, Wisconsin, USA), as described by the manufacturer's protocol. In some experiments, 1 and 100 ng/mL LPS (Sigma-Aldrich) were employed to treat the THP-1 cells. Eight hours after medium replacement, the concentrations of IL-1β, IL-6, IL-8 and TNF-α in culture supernatants were measured by ELISA (BD Biosciences). Final concentrations were calculated by normalisation with the activity of the Renilla cotransfected reporter vector (pGL4.74[hRluc/TK]).
4
Measuring NF-κB Activity and Luciferase Burden
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For assessing NF-κB activity, cells were cotransfected with 4× NF-κB-Luc, provided by Johannes A. Schmid (Addgene; plasmid no. 111216), and pGL4.74-hRluc/TK (Promega) using the transfection reagent FuGene HD (Promega). Cells were treated with TNF-α (100 ng/mL) for 24 h and lysed using passive lysis 5× buffer (Promega). Cell extracts were prepared for measuring luciferase activity using the dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega). For assessing burden with luciferase-expressing strains, at 24 hpi, cells were lysed with passive lysis 5× buffer (Promega) followed by addition of luciferin for measuring luciferase activity. All measurements were done with the EnSpire 2300 multilabel reader (PerkinElmer).
5
MMP9 Promoter Cloning and Activity Assay
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Linked to the luciferase gene, the different promoter fragments of MMP9 were cloned in the pGL4.16 (luc2/Neo) vector (Promega, Fitchburg, WI) according to the manufacturer’s instructions. The control vector was pGL4.74 (hRluc/TK) (Promega). The COLXVI cells were transiently transfected with FuGene HD Transfection Reagent (Roche) with the described vectors. The luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufactureŕs instructions. To design the cloning promoters the sequence of the MMP9 transcript ENST00000372330 from Ensembl.org (accessed 2013 Dec 20) was used. Promoter primers: 90-bp promoter: 5′-TACATTTACATTGGTACCAGCACTTGCCTGTCAAGGA-3′ and 5′- TTGATACTCGAGCCAGCACCAGGAGCACC-3′ ; 530-bp promoter: 5′-TACATTGGTACCAAAGAGGACAGAGCCTGGA-3′ and 5′- TTGATACTCGAGCCAGCACCAGGAGCACC-3′ ; 930-bp promoter: 5-‘tacattGGTACCTCTTGGGTCTTGGCCTTAGT-3′ and 5′- TTGATACTCGAGCCAGCACCAGGAGCACC-3′ .
6
Influenza Polymerase Activity Assay
7
Luciferase Assays in HEK293T Cells
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HEK293T cells were maintained in DMEM High Glucose, 1 mM Na pyruvate, 6 mM L-glutamine, 10% fetal bovine serum. For the luciferase assays HEK293T cells were seeded into 96-well plates (5000 cells/well) and transfected 18 h later with X-tremeGENETM HP DNA transfection reagent (Roche). The plasmids listed in Supplementary Table 2 were transfected as follows: pGL4.74(hRluc/TK) (Promega): 1 ng, luciferase reporter constructs: 40 ng, CMV:huΔβ-Cat: 10 ng, Sp5 expression constructs: 20 ng, huWnt3 and huLRP6: 40 ng. Total DNA amount was adjusted with pTZ18R to 100 ng per well. To measure Firefly and Renilla luciferase activities, the samples were prepared using the Dual-Luciferase Reporter Assay System (Promega), transferred to a white OptiPlateTM-96 (PerkinElmer) and measured with a multilabel detection platform (CHAMELEONTM).
8
NF-κB Transcriptional Activity Assay
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Double-strand DNA containing 5×NF-κB RE (5× 5′-GGGGACTTTCC-3′) or 5×mutated NF-κB RE (5× 5′-GGTTACTTTAA-3′) was inserted into NheI–HindIII site of pGL4.23 (Promega). HaCaT-mock and HaCaT-BLT2 cells were seeded onto 12-well plates at a density of 1.75 × 105 cells/well in DMEM containing 10% FCS and cultured for 24 h. The cells were washed twice with FCS-free DMEM, followed by the addition of 1 ml DMEM containing 10% charcoal-treated FCS. After 2 h, the cells were co-transfected with pGL4.23-5×NF-κB RE or pGL4.23-5×mutated NF-κB RE and pGL4.74 [hRluc/TK] (Promega) using Lipofectamine LTX (Life Technologies). 18 h later, the cells were stimulated with 1 µM 12-HHT for another 6 h. Lysates were prepared by adding 200 µl lysis buffer (Tokyo Ink), and firefly and Renilla (sea pansy) luciferase activities were measured with the PicaGene Dual Sea Pansy Luminescence kit (Tokyo Ink).
9
Synthesis and Characterization of AGF94
10
Cloning and Validation of Promoter Constructs
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The reporter plasmids pGL4.10[luc2] and pGL4.74[hRluc/TK] were purchased from Promega (Madison, WI, USA). The pGL4.74[hRluc/TK] which encodes Renilla luciferase was used as an internal control for transfection efficiency. The 2,000-bp upstream region and the 50-bp downstream region of the Dhh transcription start site10 (link) were amplified with KOD Fx, rDhh_-2000F_SacI (5′-TCCGAGCTC ctgagcaagccatgaggagca-3′; the SacI site is underlined) and rDhh_+50R_BglII (5′-GAAGATCT ggtttctgctgcccagctccgg-3′; the BglII site is underlined). The PCR products were cloned into pGL4.10[luc2] (Dhh_−2000/+50_pGL4.10).
The expression, pCMV6-Entry, rGATA4-pCMV6 and rGATA6-pCMV6 vectors were purchased from Origen (Rockville, MD, USA). Rat Gata4 and Gata6 were subcloned into pCMV6-Entry (rGATA4-pCMV6) and pCMV6-Entry (rGATA6-pCMV6), respectively.
All constructs were confirmed to have no mutation, no insertion, and no deletion by sequencing analysis with a BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Both strands were read with sequence primers.
The expression, pCMV6-Entry, rGATA4-pCMV6 and rGATA6-pCMV6 vectors were purchased from Origen (Rockville, MD, USA). Rat Gata4 and Gata6 were subcloned into pCMV6-Entry (rGATA4-pCMV6) and pCMV6-Entry (rGATA6-pCMV6), respectively.
All constructs were confirmed to have no mutation, no insertion, and no deletion by sequencing analysis with a BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Both strands were read with sequence primers.
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