RAW 264.7 cells were co-transfected with pNF-κB-Luc plasmid and pRL-TK vector (Promega, Madison, WI) using Lipofectamine 3000 reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instruction. After 24 h of transfection, cells were pretreated with 2′-O-GH for 2 h and then stimulated with LPS (100 ng/mL) for another 12 h. Cells were lysed and luciferase activities were performed using the dual-luciferase reporter assay kit (Promega).
Pnf κb luc plasmid
Manufactured by Promega
The PNF-κB-luc plasmid is a DNA construct that contains the firefly luciferase gene under the control of the NF-κB regulatory element. It is designed to measure NF-κB transcriptional activity in cells.
Automatically generated - may contain errors
Lab products found in correlation
Prl tk renilla reference plasmid, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Luciferase reporter assay system, by Promega (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Prl tk vector, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Rg027, by Beyotime (1 mentions)
7 protocols using pnf κb luc plasmid
1
NF-κB Activation Assay in RAW 264.7 Cells
2
Evaluating NF-κB Activation by EDA Proteins
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HEK293T cells stably expressing full-length EDAR or HaCaT cells were seeded in 12-well dishes one day before transfection. 500 ng of pNF-κB Luc plasmid (Promega, E8491), and 10 ng of pRL‐TK Renilla reference plasmid (Promega, E2231) were co-transfected for each well using Lipofectamine 3000 (Invitrogen). Twenty-four hours after the transfection, an equivalent volume of the supernatant of WT or mutated EDA· A1THD proteins were added to each well, respectively. After 12 h (for HEK293T) or 18 h (for HaCaT), Firefly luciferase activity in the cell lysates was measured and normalized to Renilla luciferase activity using a dual-luciferase reporter assay system (Promega).
3
NF-κB Activation by P. aeruginosa
4
Assessing NF-κB Activation by Luciferase Assay
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AnxA2 siRNA transfection was performed as described previously. AnxA2 knock-down or normal MH-S cells were transiently transfected with pNF-κB-luc plasmid (Promega) for 24 h, then stimulated with 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO) in RPMI 1640 medium for another 12 h. Cell lysates were subjected to luciferase activity analysis using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instruction47 (link). Firefly luciferase and renilla luciferase activity were also measured by Xenogen IVIS optical imaging system (Caliper Life Sciences).
5
Assessing NF-κB Pathway Activation
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Stable HEK293T‐EDAR cells were seeded in 12‐wells plates dishes the day before transfection. Five hundred nanograms of pNF‐κB Luc plasmid (Promega), 10 ng of pRL‐TK Renilla reference plasmid (Promega), and the expression plasmid containing either wild‐type EDA1 or the mutated EDA1 were co‐transfected into each well using Lipofectamine 2000. Twenty‐four hours after the transfection, firefly luciferase activity in the cell lysates was measured and normalized with renilla luciferase activity using the dual luciferase assay system (Promega). Each experiment was performed in triplicate and repeated three times. Data were assessed by Student’s t test (p < .05).
6
NF-κB Activation by P. aeruginosa
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Transient transfections were performed with 2 × 105 MLE-12 cells plated in 6-well plates by using pNF-κB-luc plasmid (Promega, Madison WI) following the manufacturer’s instruction. 24 h after transfection, the cells were infected with PAO1 for 12 h. Cell lysates were subjected to luciferase activity analysis by using the Luciferase Reporter Assay System (Promega).
7
Plasmid Transfection and Luciferase Assay
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The pEGFP-N1-LjRIPK1a and pcDNA3.1-HA-LjTRAF3a/6 plasmids were constructed and extracted using the TAKARA MidiBEST Endo-free Plasmid Purification Kit, and the concentration was determined to be more than 0.5 μg/μL. HEK293T cells (1 × 106 cell/mL) were plated in 96-well plates and transfected 24 h later with a mixture of DNA by using Lipofectamine 3000 (Invitrogen). The mixed DNA contained 10 ng/well RL-TK vector, 100 ng/well NF-κB or ISRE vector and complementary empty vectors. The pNF-κB-Luc plasmid, pISRE-Luc plasmid, and pRL-TK-luc plasmid were purchased from Promega. The empty vector was used as the negative control group, and the positive control group used the immunomodulator concentrations as follows: LPS, 2 μg/μL; TNFα, 2 μg/μL; and IFN, 2 μg/μL. After 24 h of transfection, the immune modulator concentration was added for stimulation for 6 h. Then, samples were detected using SpectraMax i3 using 560 and 465 nm wavelengths, respectively, according to the manufacturer's instructions (Beyotime, RG027).
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