Figure 1

Outline of the dual platform approach used to profile NF-κB family dimers. Double-purified, His-tagged NF-κB dimers interact with DNA-probes (microarray) or DNA-ligands (electrophoretic mobility shift assay-sequencing (EMSA-Seq)). Two separate stains are available for the visualization of DNA and protein on EMSA-gels. SYBR Green highlights both DNA bound by the dimer ('bound DNA') and also unbound DNA ('free DNA'). The SYPRO Ruby stain identifies proteins such as those within a dimer-DNA complex ('complex'). Both microarray and EMSA-Seq platforms generate data that provide binding affinities for individual sequences that interact with a dimer. Profiles of nine different dimers illustrating their binding affinities for 803 sequences were constructed using microarrays. In addition, RELARELA, RELAp50 and RELAp52 were also profiled using EMSA-Seq. Deep sequencing revealed dimer-specific binding affinities for distinctive groups of 11-mer sequences. Two classes of these sequences, formed on the basis of similarity to a reference NF-κB binding-model, were used as targets for a UV footprinting experiment. Finally, differences for in vitro binding potential as determined using binding affinities from EMSA-Seq and differences for in vivo binding as established by a ChIP-Seq study were then co-examined across 7,762 comparisons of paired individuals.