Figure 2

Experimental design of the site-specific ChIP. Schematic illustration of the strategy used for the analysis of individual TFBSs (here Sp1). Immunoprecipitation and isolation of occupied binding motifs was enabled by specific enzyme digestion on positions flanking the TFBSs. Enzyme cutting sites of the respective endonucleases (RsaI, BfaI and SacI) are depicted by dashed lines. Immunoprecipitated DNA was analyzed by PCR using two sets of primer assays; one for the specific detection of allocated Sp1 TFBSs and a control set for the determination of the efficiency of the enzyme digestion. Sp1 TFBSs within egfr promoter region are represented by grey oval disks, indicating their positions in relation to the translational start codon (ATG). Black bars depict the targeted egfr regions of the enzyme digestion control primer assays. Signals appearing in the enzyme digestion control PCR indicate a failed fragmentation. The grey bars denote location and length of PCR products of primers used for the specific detection of immunoprecipitated DNA fragments containing the targeted Sp1 binding sites (a, b, c, d). Signals indicate binding sites with bound TF (here Sp1).