Protein-ligand binding sites were retrieved from the 2006 release of the sc-PDB (http://bioinfo-pharma.u-strasbg.fr/scPDB), a database of 6 415 druggable protein-ligand binding sites [39] from the Protein Data Bank.
The chromosomal locations of AGO2 binding sites were retrieved from the CLIPZ database [68], which releases RNA-binding protein (RBP) binding site data generated by cross-linking and immunoprecipitation (CLIP) mapping technique.
Sites within 100 bp of predicted NR2E1 binding sites were retrieved from the oPOSSUM database.
Predicted transcription factor binding sites were retrieved from MSigDB (Subramanian et al., 2005; Matys et al., 2006).
Experimentally derived transcription factor binding sites were retrieved from TRANSFAC database 6.0 [ 24], the eukaryotic transcription factors database.
For each of the identified specific motifs an initial list of 30 to 100 putative binding sites was retrieved and the list was reduced by application of a distance and similarity criterion to yield a few putative highly specific binding sites per TF (visualized in Figure 3; data in Additional file 8).
Experimentally characterized human transcription factor binding sites (TFBS) were retrieved from the Professional release 11.3 (9/10/07) of the TRANSFAC database [ 18].
In addition, consensus sequences of TF binding sites (TFBS) were retrieved from two publicly accessible comprehensive plant cis-element databases, PLACE [ 11] and AtcisDB [ 12].
After the antigen was retrieved in a citrate buffer (0.01 M, pH 6.0) and subjected to heat treatment using a microwave, the nonspecific binding sites were blocked with 10% non-immune goat serum for 30 minutes.
The underlying assumption is that true binding sites will be retrieved in both replicate datasets within a similar confidence ranking, while low confidence peaks, which are expected to contain also false positives, will show lower consistency in the reproducibility assay.
Expression levels of eight transcription factors targeting on 3 identified binding sites can be retrieved from Oncomine, where six proteins, JUN, JUNB, JUND, FOS, FOSL1, and FOSL2 bind on AP-1 site, EGR1 (early growth response element 1) binds on KROX site, and STAT3 (signal transducer and activators of transcription 3) binds on STAT3 site.
