HaCaT cells were transiently transfected with TGFβ/Smad-responsive promoter-reporter pCAGA12-MLP-luc for 36–48 h prior to stimulation with TGFβ. pCMV-β-gal or pEGFP were co-transfected as controls for normalization. Additional constructs or siRNAs were included in the transfections according to the figures. Luciferase reporter assays were performed with the enhanced luciferase assay kit from BD PharMingen, Inc. (BD Biosciences, Stockholm, Sweden), according to the protocol of the manufacturer. Normalized promoter activity data are plotted in bar graphs that represent average values from triplicate determinations with standard deviations. Each independent experiment was repeated at least twice.
Enhanced luciferase assay kit
Manufactured by BD
Sourced in United States
The Enhanced Luciferase Assay Kit is a laboratory equipment product that facilitates the detection and quantification of luciferase enzyme activity. The kit provides the necessary reagents and components to perform luciferase-based assays, a widely used technique in various research applications.
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9 protocols using enhanced luciferase assay kit
1
TGFβ-Induced Luciferase Reporter Assay
2
Measuring TGFβ-Induced Transcriptional Activity
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PC3U cells were transiently transfected using TransIT-X2® reagent with TGFβ/Smad-responsive promoter-reporter pCAGA12-MLP-luc for 24 h prior to stimulation with TGFβ. pCMV-β-gal was co-transfected as control for normalization. Additional constructs or siRNAs were included in the transfections according to the figures. All cells were lysed in lysis buffer containing 5 mM Tris-phosphate buffer, pH 7.8, 2 mM dithiothreitol (DTT), 2 mM trans-1,2-diaminocyclohexane-N,N,N′,N′-tetra-acetic acid, 5% glycerol, and 1% Triton X-100. The β-galactosidase assay was performed by mixing the cell lysate with 100 mM sodium phosphate, pH 7.3, 1 mM MgCl2, 50 mM β-mercaptoethanol, and 0.67 mg/ml of o-nitrophenyl β-d-galactopyranoside, and the absorbance was monitored at 420 nm. Luciferase reporter assays were performed with the enhanced luciferase assay kit from BD PharMingen, Inc. (BD Biosciences, Stockholm, Sweden), according to the protocol of the manufacturer. Normalized promoter activity data are plotted in bar graphs that represent average values from triplicate determinations with standard deviations. Each independent experiment was repeated at least three times.
3
Luciferase reporter assay for TGF-β signaling
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Cells that were 50% confluent in 12-well dishes were transfected with one of the SBE-4-Luc, 3TP- Luc, SMAD3-dependent (CAGA)12-Luc reporter plasmids using Lipofectamine 2000 (Invitrogen). The cell extracts were prepared 48 h after transfection, and the luciferase activity was quantified using the Enhanced Luciferase Assay Kit (BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.
4
BRE-Luciferase Transfection and Activity Assay
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3 × 104 cells were transfected with a BRE-Luciferase reporter plasmid using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). The cell lysates collected 48 hr after transfection, and the luciferase activities were measured using the Enhanced Luciferase Assay Kit (BD Biosciences, San Jose, CA, USA).
5
STAT3 and CD133 Reporter Assay
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Reporter activities were determined as previously described [51 (link)]. 1 × 105 cells per well in 12-well dishes were seeded and cotransfected with 0.5 μg of STAT3 or CD133 reporter gene constructs and 0.5ug of pCMV-β-gal using Lipofectamine 2000 (Invitrogen). The cell lysates were prepared 48 hr after transfection. The luciferase activities were measured with the Enhanced Luciferase Assay Kit (BD Biosciences).
6
Transcriptional Regulation Assay
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Cells that were 50% confluent in 12-well dishes were transfected using Lipofectamine 2000 (Invitrogen). A total of 0.5 μg E-cadherin, Twist-1, Twist-2, or STAT3 reporter gene constructs and 0.5 μg of pCMV-β-gal were cotransfected per well. The cell extracts were prepared 48 hrs after transfection, and luciferase activity was quantified using the Enhanced Luciferase Assay Kit (BD Biosciences). All of the experiments were performed in triplicate.
7
Luciferase Reporter Assays for STAT3 and CD133
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Luciferase reporter assays were performed as previously described [24 (link)]. 1 × 104 cells were seeded in 12-well dishes were transfected using Lipofectamine 2000 (Invitrogen). A total of 0.5 μg pGL3-STAT3 or pGL3-CD133 reporter gene constructs and 0.5 μg of pCMV-β-gal were co-transfected per well. The cell extracts were prepared 48 h after transfection, and the luciferase activity was quantified using the Enhanced Luciferase Assay Kit (BD Biosciences). All experiments were performed in triplicate. The human STAT3 promoter (− 1555/ + 133) (Sequence ID: NM_213662) and human CD133 promoter (− 1313/− 1) (Sequence ID: NM_001145847) were cloned into the pGL3 Vector (Figure S1).
8
Measuring Thyroid Hormone Receptor Binding
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Thyroid hormone receptor binding activity was measured using a GAL4 system, which comprised a plasmid expressing the fusion protein of the ligand-binding domain of TRα and the DNA-binding domain of GAL4. To estimate TH promoter activity, we inserted a 6.0-kb fragment of the rat TH promoter upstream of the luciferase gene obtained from the pGL3-basic plasmid37 (link). A pRSV-Renilla-luciferase plasmid expressing Renilla luciferase was used for normalization. On differentiation day 6, the cells were transfected with plasmids using Lipofectamine 2000. The following day, the cells were harvested and lysed for 10 min using cell lysis buffer (BD Pharmingen, San Diego, CA). The cell lysates were mixed with Renilla luciferase reagent (Enhanced Luciferase Assay kit, BD Pharmingen). Luciferase activity was measured using a luminometer (Berthold Detection Systems, Huntsville, AL, USA).
9
Measuring BRE-Luciferase Reporter Activity
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3 10 4 cells were transfected BRE-Luciferase reporter plasmid using Lipofectamine 2000 (Invitrogen). The cell lysates collected 48 hr after transfection, and the luciferase activities were measured using the Enhanced Luciferase Assay Kit (BD Biosciences).
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