PTECs were cultured in 3.5 cm culture dishes and transfected after 24 h with 4 µg SMAD3-SMAD4 transcriptional reporter (CAGA12-Luc) [37 (link)] and 200 ng renilla luciferase reporter as a transfection control (pGL4.75[hRlucCMV]; Promega; #E6931) using 10 µl Lipofectamine 2000 according to the manufacturer’s protocol (Life Technologies; #11668019). Cells were maintained under serum-free conditions from the moment of transfection and fluid flow was started 24 h after transfection. Cells were lysed after 20 h of fluid flow stimulation using a cone–plate device. Firefly and renilla luciferase activities were measured on a luminometer (Victor 3; PerkinElmer) using the Dual-Luciferase Reporter Assay System (#E1960) from Promega according to the manufacturer’s instructions. Firefly luminescence was corrected for renilla to get the relative activity of the reporter.
Pgl4.75 hrluc cmv
Manufactured by Promega
The PGL4.75 hRluc/CMV is a luciferase reporter vector that contains the human codon-optimized Renilla luciferase (hRluc) gene under the control of the cytomegalovirus (CMV) promoter. This vector is designed for use in luciferase-based gene expression assays.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Dual glo luciferase assay system, by Promega (3 mentions)
Dual luciferase reporter assay, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Victor3, by PerkinElmer (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
Pncmo2, by Takara Bio (1 mentions)
Rosiglitazone, by Merck Group (1 mentions)
Ppre x3 tk luc, by Addgene (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Passive lysis buffer (plb), by Biotium (1 mentions)
Firefly and renilla dual luciferase assay kit, by Biotium (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
Pmirglo vector, by Promega (1 mentions)
Dual glo luciferase reagent, by Promega (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Lysis buffer, by Promega (1 mentions)
M50 super 8x topflash, by Addgene (1 mentions)
19 protocols using pgl4.75 hrluc cmv
1
Transcriptional Reporter Assay for SMAD3-SMAD4 Signaling
2
Investigating Antiviral Immune Signaling
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Mouse monoclonal Flag tag antibody was from Sigma (USA); rabbit polyclonal HA tag antibody, rabbit polyclonal ARIH1 antibody and rabbit polyclonal IRF3 antibody were from ABclonal Biotechnology (China); rabbit polyclonal phosphorylated IRF3 (p-IRF3) antibody was from Abcam (USA); mouse monoclonal Myc tag antibody, rabbit polyclonal NP and M1 of H1N1/PR8 antibody and rabbit polyclonal RIG-I antibody were from Proteintech (Wuhan, China); mouse polyclonal GAPDH antibody were from BioPM (Wuhan, China); horseradish peroxidase (HRP) conjugated secondary antibodies were from Beyotime Biotechnology (China).
ARIH1, RIG-I, RIG-I-N (The N-terminal CARD domain of RIG-I), VISA, TBK1, and SQSTM1 expression plasimds; a luciferase reporter plasmid for the IFN-β promoter (pIFN-β-Luc); and a luciferase reporter plasmid pRNP-Luc with a pol I transcription unit were constructed by our laboratory. A Renilla control plasmid (pGL4.75 hRluc/CMV) was purchased from Promega. The PHW-PR8-PB2, PB1, PA, and NP plasmids were constructed from H1N1/PR8 as previous described [20 (link)]. Myc-tag ubiquitin plasmid (pMyc-UbWT), Myc-tag ubiquitin mutant in which all lysine residues except K63 were mutated to arginine plasmid (pMyc-UbK63), and Myc-tag mutant in which only the K63 residue was mutated to arginine plasmid (pMyc-UbK63R) were get from Hedgehogbio (Shanghai, China).
ARIH1, RIG-I, RIG-I-N (The N-terminal CARD domain of RIG-I), VISA, TBK1, and SQSTM1 expression plasimds; a luciferase reporter plasmid for the IFN-β promoter (pIFN-β-Luc); and a luciferase reporter plasmid pRNP-Luc with a pol I transcription unit were constructed by our laboratory. A Renilla control plasmid (pGL4.75 hRluc/CMV) was purchased from Promega. The PHW-PR8-PB2, PB1, PA, and NP plasmids were constructed from H1N1/PR8 as previous described [20 (link)]. Myc-tag ubiquitin plasmid (pMyc-UbWT), Myc-tag ubiquitin mutant in which all lysine residues except K63 were mutated to arginine plasmid (pMyc-UbK63), and Myc-tag mutant in which only the K63 residue was mutated to arginine plasmid (pMyc-UbK63R) were get from Hedgehogbio (Shanghai, China).
3
Dual-Luciferase Assay of Influenza Polymerase
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A dual-luciferase reporter assay system (Promega) was used to measure polymerase activities. To generate each of the 16 possible RNP complex combinations, 0.5 μg of the luciferase report plasmid pPoll-NP-Luc (pNP-Luc), which was kindly provided by Dr. Hualan Chen (Harbin Veterinary Research Institute), and 20 ng of the internal control plasmid Renilla (pGL4.75 hRluc/CMV) (Promega) were co-transfected into 293T cells together with 0.5 μg each of plasmid PHW2000 expressing PB2, PB1, PA, or NP derived from HM/06 or LN/09. Cells were lysed 36 h after transfection, and firefly and Renilla luciferase activities were measured in accordance with the manufacturer’s instructions. Values were reported as the results of three independent experiments and were standardized to the activities of HM/06-derived polymerase.
4
Nuclear Receptor Expression and Reporter Assays
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Expression plasmids encoding human nuclear receptors CAR1 and CAR3 [67 (link)] and RXRα [68 (link)] and VDR [69 (link)] have all been described previously.
Expression plasmids encoding fusion proteins of GAL4-DNA binding domain (DBD) and the receptor interaction domains (RID) of nuclear receptor co-activator (NCOA) 1 (residues 583-783) [70 (link)], nuclear receptor co-repressor (NCOR) 2 (residues 1109-1330), or PXR ligand binding domain (LBD) helix 1 part (residues 132-188), as well as expression plasmids encoding fusion proteins of the VP16 activation domain (AD) and the whole (residues 108-434) or part (189-434) of the PXR-LBD [69 (link)] have been described earlier.
The following firefly luciferase reporter gene plasmids were used: CYP3A4 enhancer/promoter reporter gene plasmid pGL4-CYP3A4(7830Δ7208-364) [71 (link)]; CYP2B6 enhancer/promoter reporter gene plasmid pB-1.6k/PB/XREM [72 (link)]; pGL3(DR3)3Tk, with a trimer of CYP3A23 direct repeat (DR) 3 motif [73 (link)]; GAL4-dependent pGL3-G5 [70 (link)].
For normalization of transfections, Renilla luciferase expression plasmid pGL4.75[hRLuc/CMV] (Promega) was used.
Expression plasmids encoding fusion proteins of GAL4-DNA binding domain (DBD) and the receptor interaction domains (RID) of nuclear receptor co-activator (NCOA) 1 (residues 583-783) [70 (link)], nuclear receptor co-repressor (NCOR) 2 (residues 1109-1330), or PXR ligand binding domain (LBD) helix 1 part (residues 132-188), as well as expression plasmids encoding fusion proteins of the VP16 activation domain (AD) and the whole (residues 108-434) or part (189-434) of the PXR-LBD [69 (link)] have been described earlier.
The following firefly luciferase reporter gene plasmids were used: CYP3A4 enhancer/promoter reporter gene plasmid pGL4-CYP3A4(7830Δ7208-364) [71 (link)]; CYP2B6 enhancer/promoter reporter gene plasmid pB-1.6k/PB/XREM [72 (link)]; pGL3(DR3)3Tk, with a trimer of CYP3A23 direct repeat (DR) 3 motif [73 (link)]; GAL4-dependent pGL3-G5 [70 (link)].
For normalization of transfections, Renilla luciferase expression plasmid pGL4.75[hRLuc/CMV] (Promega) was used.
5
Plasmid Vectors for Cellular Assays
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p3×FLAG-CMV-9 and p3×FLAG-CMV-10 vectors were purchased from MilliporeSigma. The pAP1(1)-Luc vector was purchased from Affymetrix, Inc.; pGL4.75[hRluc CMV] was from Promega Corporation; and pNCMO2 was from Takara Bio, Inc.
6
Transcriptional Regulation of PPARs
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To investigate the transcriptional activity of PPARs, we used a peroxisome proliferator response element (PPRE)-driven luciferase reporter gene approach (Kim et al., 1998 (link)). In brief, CHO cell lines were transfected with a reporter construct containing three copies of the PPRE placed upstream of the thymidine kinase promoter- firefly luciferase fusion gene (PPRE X3-TK-luc, Addgene plasmid #1015 ). In addition, the cells were transfected with a cytomegalovirus promoter-driven Renilla luciferase reporter gene (pGL4.75 hRluc/CMV, Promega, E6931) used as transfection control. Where indicated, transfectants were treated with 5 µM of rosiglitazone (Sigma, R2408) or with conditioned medium containing 5 µg/ml of human apoE3 or apoE4 for 24 h. At 24 to 48 h after transfection, cells were lysed in buffer (50 mM Tris-HCl, pH 7.4, 140 mM NaCl and 1% Triton X-100) and lysates used to assay luciferase activity in 96-well plates using the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer's instructions. The luminescence-based PPAR activity (firefly luciferase) was normalized to the internal Renilla luciferase activity.
7
Functional Assessment of CEACAM21 Promoter Variants
8
Luciferase Assay for IL-23 Binding Site
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The sequence of the putative PRINS binding site of IL-23 cDNA that was identified in silico was synthesized and inserted into the pmirGLO vector (Promega, Madison, WI, USA) between the Dra I and Xba I restriction sites, resulting in the pmirGLO-IL23BS construct (Supplementary Figure S4 ). HEK293 cells were transiently transfected by the PRINSpcDNA3.1 or pmirGLO plasmid as controls. Cells were also co-transfected with 0.5 μg pmirGLO-IL23BS or empty pmirGLO plasmid together with the PRINSpcDNA3.1 vector. In each transfection experiment, 0.025 μg renilla-luciferase-expressing pGL4.75 hRluc/CMV (Promega) plasmid was used as internal control. Cells were harvested after 24 h, washed with PBS (phosphate-buffered saline) and lysed with passive lysis buffer (Biotium, Hayward, CA, USA), and luciferase activities were measured using the Firefly and Renilla Dual Luciferase Assay Kit (Biotium) and SYNERGY/HTX Multi-Mode reader (Bio Tek Instruments, Winooski, VT, USA), according to the manufacturer’s instructions. The luciferase activity derived from the pmirGLO plasmids was normalized to the renilla-luciferase activity.
9
Dual-Luciferase Assay in Hepatocytes
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Approximately 1.0–1.5 × 105 isolated primary hepatocytes were co-transfected per well in 24-well plates with 1.5 µg of promoter reporter construct and 0.5 µg of the reference reporter construct, pGL4.75[hRluc/CMV] (Promega), encoding Renilla luciferase. Transfection was performed using electroporation program P16 (Table 1 ). At 24 hours post-transfection, fresh complete L15 medium (L15 GlutaMAX, 5% fetal bovine serum, 1× streptomycin–penicillin) was added to transfected cells and incubated at 15°C under atmospheric conditions for an additional 24 hours. To quantify firefly and Renilla luciferase activities, medium on cells was replaced with 100 µl of Dulbecco's modified Eagle's medium (Sigma) and 100 µl Dual-Glo Luciferase reagent (Promega) per well and incubated for approximately 30 minutes. Luminescence was read on a Synergy H1 Hybrid multi-mode microplate reader (BioTek). Luminescence from Renilla luciferase activities was measured 10 minutes after adding 100 µl of Dual-Glo Stop & Glo reagent. Firefly luminescence was normalized to Renilla luciferase luminescence.
10
Quantifying Transcriptional Activity via Luciferase Assays
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The following plasmids were used for luciferase reporter assays: TEAD‐reporter construct (8xGTIIC-luciferase, Addgene, Plasmid #34615), TOP‐reporter construct (M50 Super 8x TOPFlash, Addgene, Plasmid #12456) and CMV-Renilla (pGL4.75[hRluc/CMV], Promega). CAFs were seeded in 6-wells plates at 70% confluency. Cells were transfected with 2 µg TOPFlash or TEAD‐reporter and 100 ng of CMV-Renilla cDNA constructs using Lipofectamine 3000 according to the manufacturer’s instructions. Cells were lysed 48 h after transfection in 100 μL of lysis buffer (Promega). Aliquots of the cell lysates were used to read luciferase emission using Dual-Glo® Luciferase Assay System (Promega) according to the manufacturer’s instructions. Luciferase activities were normalised to Renilla activity.
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