Cells were co-transfected with:750 ng effector plasmid (pM3-based; see above), 10 ng Renilla luciferase reporter plasmid (hRLuc/Tk; Promega # E692A, and 250 ng firefly luciferase reporter plasmid that contains five GAL4 DNA binding site upstream of a strong SV40 promoter (pGl3control-5’Gal4 [48 (link)];). Cells were subjected 24-h post-transfection to a dual luciferase assay (Promega) following the manufacturer’s protocol and using a Berthold single channel luminometer (Lumat LB9501). Firefly luciferase activities were normalized with respective Renilla luciferase activities for each sample. The luciferase activity in the presence of the effector plasmid expressing the GAL4 DNA binding domain alone represents the reference transcriptional capacity. Fold repression was calculated experiment-wise by dividing the normalized luciferase activity of that reference through normalized luciferase activities of the sample with the respective test effector plasmid. Note that identical results for certain constructs are presented in more than one figure for ease of comparison.
Hrluc tk
Manufactured by Promega
Sourced in United States
The HRluc/TK is a dual reporter system that contains the Renilla luciferase (hRluc) and the herpes simplex virus thymidine kinase (HSV-TK) reporter genes. This system allows for the detection of gene expression and cell viability in a single assay.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Luc2p nf κb re hygro, by Promega (2 mentions)
Dual luciferase assay, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
X tremegene hp reagent, by Roche (1 mentions)
Dual luminescence assay kit, by GeneCopoeia (1 mentions)
Pgl4.33 luc2p sre hygro, by Promega (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Penicillin streptomycin amphotericin b suspension, by Fujifilm (1 mentions)
Prl tk, by Promega (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Fugene transfection reagent, by Promega (1 mentions)
Control rna, by GenePharma (1 mentions)
9 protocols using hrluc tk
1
Dual Luciferase Assay for Transcriptional Regulation
2
Cardiac Transcription Factor Promoter Profiling
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The TNNT2 (HPRM12846-PG04) and NPPA (natriuretic peptide A; HPRM23486-PG04) promoter activities were measured using a Dual Luminescence Assay Kit with a dual reporter construct containing Gaussia luciferase (GLuc) and secreted alkaline phosphatase (SEAP) side by side from a single sample (all from GeneCopoeia, Inc.). Each promoter is placed upstream of the GLuc reporter gene and contains a specific cardiac transcription factor as an insert. A secondary reporter gene SEAP was used to monitor the transfection efficiency for normalization. To detect serum response element (SRE) promoter activities, both SRE fragment (pGL4.33 [luc2P/SRE/Hygro], Promega) and pGL4.74 (hRluc/TK, Promega) were co-transfected into human cells by using X-tremeGene HP reagent (Roche). The relative expression of SRE (luc2P) construct was normalized using a control vector (hRluc) introduced into the same cells. Human cells were transfected with human NKX2-5 cDNA (SC122678), human NKX2-5 shRNA (short hairpin RNA; TR311165B), human HAND1 cDNA (SC122690), human HAND1 shRNA (TR316857C), human NOTCH1 cDNA (SC308883), or human NOTCH1 shRNA (TR302916D) along with single- or dual-reporter constructs (all from OriGene Technologies, Inc.). The luciferase activities were measured by the Glomax-Multi+Detection System (Promega) 48 hours after transfection.
3
Dual-Luciferase Assay for NQO1 ARE Activation
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For the dual-luciferase reporter gene assay, HEK293T cells were transfected with the NQO1 ARE-luciferase plasmid along with the Renilla luciferase expression plasmid pGL4.74 (hRluc/TK) (Promega) and different amounts of expression vectors for Flag-XBP1s. At 24 h post-transfection, cells were treated with a known inducer of Nrf2, tBHQ (Sigma), for 16 h. The transfected cells were then lysed with passive lysis buffer (Promega), and both firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. The experiment was repeated three times with triplicate samples, and the data are expressed as mean ± standard deviation (SD).
4
Lentiviral Plasmid Construction and Influenza Replicon Assay
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The PEZ-Lv242-TRAF3 lentiviral plasmid and its domain deletion plasmids were commercially constructed by GeneCopoeia, Guangzhou, China. The psPAX2, HA-UB, and VSVG packing plasmids of lentivirus were kept in our laboratory. Plasmids of viral ribonucleoprotein complex (vRNP) subunits including pHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2KPB2, and pPolI-Fluc (firefly luciferase reporter plasmid) used in the mini-replicon system were kindly presented by Professor Bojian Zheng (University of Hong Kong, HK, China). The IgK-IFN-luc plasmid was bought from Miaolingbio, Wuhan, China. The reference plasmid hRluc-TK was obtained from Promega Corporation (Madison, WI, USA).
5
Luciferase Assay for Gene Regulation
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The luciferase reporter constructs containing the cis-elements in the first intron of vas described above were used for luciferase reporter assays. The hRluc/TK (Promega, pGL4.74) served as an internal control. About 50 ng of a luc reporter construct and 1 ng of an internal control were co-transfected into S2 cells (3 × 104 cells) with either 10 ng of pAC-MamoAF-FLAG and/or 1 ng of pAC-OvoB and/or 1 ng of pAC-CBP and/or pBSK (for mocked transfected cells) using Effectene transfection reagent (Qiagen). The transfected cells were cultured in the culture medium supplemented with Penicillin–Streptomycin–Amphotericin B Suspension (Wako). Cells were lysed after 1 day of transfection with a passive lysis buffer (Promega) and luciferase activity was measured using a Dual-Luciferase reporter assay system (Promega). All experiments were carried out in triplicate.
6
Dual-Luciferase Reporter Assay
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Cells were transfected with either a TonE-driven Photinus luciferase plasmid or a κB-driven luciferase plasmid in pGL4.74 (hRluc/TK, Promega). The Renilla luciferase reporter plasmid (pRL-TK, Promega) was used as a control for transfection efficiency. Luciferase activity after 8 h of stimulation was measured using the Dual-Luciferase Assay System (Promega) according to the manufacturer’s instructions. Luciferase activity was normalized by activity of renilla luciferase.
7
Investigating NF-κB Regulation in Fibroblasts
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NPDFs were plated on 60-mm cell culture plates. NF-κB luciferase reporter gene constructs (luc2P/NF-κB-RE/Hygro and hRluc/TK; Promega, Madison, WI, USA) were transiently transfected into NPDFs by using fetal bovine serum and antibiotic-free DMEM containing 5 µL of FuGENE transfection reagent (Promega). After 5 hours of incubation, the medium was replaced with DMEM containing 10% fetal bovine serum. After incubation for 24 hours, transfected NPDFs were treated with TGF-β1 (5 ng/mL), with or without delphinidin (20 µM), MAPK inhibitors (10 µM), and NF-κB inhibitor (1 µM) at 37℃ for 2 hours. Luciferase assays were performed to determine the firefly luciferase activity relative to the Renilla luciferase activity in the cell lysate using a luminometer (Promega).
8
Mini-replicon System Plasmids
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Plasmids pHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2KPB2, and pPolI-Fluc (firefly luciferase reporter plasmid) used in Mini-replicon system were kindly presented by Professor Bojian Zheng (University of HongKong, HK, China). Renilla luciferase plasmid (hRluc-TK) was purchased from Promega (Beijing, China). The SOD3-encoding plasmid was purchased from GeneCopoeia (Guangzhou, China). The siRNA against SOD3, as well as the control RNA, were purchased from GenePharma (Shanghai, China).
9
NF-kB Activation in Nasal Fibroblasts
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Nasal fibroblasts were plated on 35-mm cell culture plates. NF-kB luciferase reporter gene constructs (luc2P/ NF-κB -RE/Hygro and hRluc/TK; Promega, Madison, WI, USA) were transfected into nasal fibroblasts using fetal bovine serum and antibiotic-free Dulbecco's Modified Eagle media containing 4 μL of purefection transfection reagent (LV750A-1, PureFection™, System Bioscience). After incubation for 20 minutes, the medium was replaced with DMEM containing 10% fetal bovine serum. After further incubation for 24 hours, the transfected nasal fibroblasts were treated with DEP (50 μg/mL). Luciferase assay was performed using a dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity, and the level of induction was reported.
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