For EMSA, IRDye 700-labelled oligonucleotides containing a consensus NF-κB binding site (5′- AGTTGAGGGGACTTTCCCAGGC-3′; LI-COR Biosciences) were incubated with nuclear extracts according to the manufacturer’s protocol (Odyssey Infrared EMSA kit). Electrophoresis of the resultant products was carried out with 5% TBE gel (Bio-Rad). Native PAGE for analysis of IRF3 dimerization was performed as previously described64 (link). Immunoblotting was carried out using standard methods. An Odyssey Imaging System (Li-COR Biosciences) was used for visualization and signal (infrared fluorescence) intensity analysis.
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NF-κB Binding Site Consensus Oligonucleotide EMSA
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Other organizations : University of North Carolina at Chapel Hill
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Variable analysis
independent variables
- Treatment with nuclear extracts
- NF-κB binding activity
- IRF3 dimerization
- Consensus NF-κB binding site oligonucleotide (5′- AGTTGAGGGGACTTTCCCAGGC-3′; LI-COR Biosciences)
- Electrophoresis conditions (5% TBE gel, Bio-Rad)
- Immunoblotting methods
- Positive control: Not specified
- Negative control: Not specified
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