Assessing NF-κB Activation by Luciferase Assay

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).

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Zhang S., Yu M., Guo Q., Li R., Li G., Tan S., Li X., Wei Y, & Wu M. (2015). Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway. Scientific Reports, 5, 15859.

Publication 2015

pNF-κB-luc plasmid LPS Dual-Luciferase Reporter Assay System

Anxa2 Assay Cell Firefly luciferase Luciferase Plasmid Promega Renilla luciferase Sirna Transfection

Corresponding Organization :

Other organizations : University of North Dakota, Sichuan University

Top 5 similar protocols

Variable analysis

independent variables

AnxA2 knock-down or normal MH-S cells

dependent variables

NF-κB transcriptional activity
Firefly luciferase activity
Renilla luciferase activity

control variables

Transfection of pNF-κB-luc plasmid
LPS stimulation (100 ng/ml)

positive controls

Normal MH-S cells transfected with pNF-κB-luc plasmid and stimulated with LPS

negative controls

AnxA2 knock-down MH-S cells transfected with pNF-κB-luc plasmid and stimulated with LPS

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