The TCF/LEF luciferase reporter plasmid, NF-κB luciferase reporter plasmid, and TK-Renilla luciferase plasmid were purchased from Promega (Madison, WI, USA). Smo plasmid was purchased from Origene (Rockville, MD, USA). The Smo mutant plasmids were generated from wild type Smo plasmid using QuickChange Site-Directed Mutagenesis kit from Agilent (Santa Clara, CA, USA) and confirmed by sequencing. Information on Smo mutants plasmids is provided in Supplementary data 2. The Gli1 plasmid and Gli2-HA plasmid were obtained from Addgene (Cambridge, MA, USA). Transient transfections were performed by Lipofectamine 2000 reagent from Invitrogen (Grand Island, NY, USA) according to the manufacturer's instructions.
Nf κb luciferase reporter plasmid
Manufactured by Promega
Sourced in United States
The NF-κB luciferase reporter plasmid is a molecular biology tool used for the detection and measurement of NF-κB transcriptional activity in cells. It contains a luciferase gene under the control of an NF-κB responsive promoter, allowing for the quantification of NF-κB activation in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Tcf lef luciferase reporter plasmid, by Promega (2 mentions)
Tk renilla luciferase plasmid, by Promega (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Flexstation 3, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Pgl3.0 vector, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Prl tk, by Promega (1 mentions)
One step cloning kit, by Vazyme (1 mentions)
7 protocols using nf κb luciferase reporter plasmid
1
Hedgehog Pathway Modulation Assay
2
NF-κB Activation by H2O2 in HeLa Cells
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HeLa cells were co-transfected with a NF-κB-luciferase reporter plasmid (Stratagene) and pRV-SV40 control plasmid for 24 h. After transfection, the cells were incubated with or without 100 μM of H2O2 for 6 h. The luciferase activities were measured by dual luciferase assay kit (Promega).
3
NF-κB Activation Assay in HEK293-hGPR43 Cells
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HEK293-hGPR43 cells were transfected with NF-κB luciferase reporter plasmid (Promega), incubated under 5% CO2 and 37°C for 24 h, and plated in 96-well white plates overnight. The next day, cells were treated with the relevant compounds for 20 min, followed by 10 ng/mL TNF-α, and incubated at 37°C for a further 6 h. Luminescence was measured using FlexStation 3 at the 15 min time-point after adding One-Glo assay reagent (Promega) to each well.
4
NF-κB Transcriptional Activity Assay
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The cells were seeded into 96-well plates at 70% confluence. After 16 h, cells were transfected with NF-κB luciferase reporter plasmid (Promega Corporation) and Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h at 37°C, cells were collected to measure the luciferase activity with the Dual-Luciferase Reporter Assay System kit (Promega Corporation). The transcriptional activities of genes were expressed as the ratio between firefly luciferase and Renilla luciferase.
5
Lentiviral-Mediated Knockdown of Sufu in NIH-3T3 Cells
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The 8 × Gli1-binding site luciferase reporter (8 × GBS-luciferase) plasmid was a kind gift from Dr. Hiroshi Sasaki. The TCF/LEF-luciferase reporter plasmid, NF-κB –luciferase reporter plasmid, and TK-Renilla luciferase plasmid were purchased from Promega (Madison, WI). The Gli2 lacking the N-termianl (Gli2ΔN) plasmid and ShhN plasmids were obtained from Addgene (Cambridge, MA). The Myc-DKK-tagged ORF clone of Homo sapiens Smo plasmid was purchased from Origene (Rockville, MD). The mutant human plasmid SmoM2 (W535L) was generated from wild type Smo plasmids using QuickChange Site-Directed Mutagenesis kit from Agilent (Santa Clara, CA) and was confirmed by sequencing. The Sufu-shRNA was purchased from Santa Cruz (Santa Cruz, CA).
Transient transfections were performed using Lipofectamine 2000 reagent from Invitrogen according to the manufacturer’s instructions. The lentiviral stocks were prepared according to previous report [19 (link)]. Briefly, the plasmid carrying the Sufu-shRNA and three packaging plasmids were co-transfected into 293T cells using Lipofectamine 2000. The viruses were harvested 24 h post transfection and 4 ml viruses were used for infected NIH-3T3 cells seeded in 10-cm dishes. Infected cells were analyzed 5–7 days post infection by western blot analyses of the expression of Sufu.
Transient transfections were performed using Lipofectamine 2000 reagent from Invitrogen according to the manufacturer’s instructions. The lentiviral stocks were prepared according to previous report [19 (link)]. Briefly, the plasmid carrying the Sufu-shRNA and three packaging plasmids were co-transfected into 293T cells using Lipofectamine 2000. The viruses were harvested 24 h post transfection and 4 ml viruses were used for infected NIH-3T3 cells seeded in 10-cm dishes. Infected cells were analyzed 5–7 days post infection by western blot analyses of the expression of Sufu.
6
NF-κB Transcriptional Activity Assay
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Daudi cells were stably infected with NF-κB Luciferase reporter plasmid (Promega, Madison, WI, USA). Equal numbers of cells were plated in 24-well plates, then treated with various concentrations of DHI for 4 h, followed by a 4-h stimulation with or without TNFα (15 ng/ml). Cells were lysed in passive lysis buffer (Promega) for 15 min. The transcriptional activity was determined by measuring the activity of firefly luciferase in a multiwell plate luminometer (Tecan, Durham, NC, USA) using a luciferase reporter assay system (Promega) according to the manufacturers’ instructions.
7
Transcriptional Regulation of Adhesion Molecules
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By using the One Step Cloning Kit (C112-02; Vazyme Biotech, Nanjing, China), NF-κB luciferase reporter plasmid, purchased from Tsingke Biotechnology (Tsingke, China), promoters of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin, were assembled into pGL3.0 vector (Promega, Madison, Wis). The aforementioned primers are listed in Table E3. HEK293 cells were transfected with an internal control plasmid (pRL-TK; Promega) and plasmids containing other promoter regions simultaneously. Luciferase activity were detected with the Dual Luciferase Reporter Assay Kit (Promega).
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