ChIP Display (CD) demonstrates a putative AR target. A) CD Gel. C4-2B cells were treated with 10 nM DHT for 4 hours to enhance AR association with target loci. Two independent AR ChIPs, and IgG control ChIPs were subjected to the CD procedure as described in Methods. In the example shown here, PCRs were performed with the 'AC' and the 'TG' PCR primers (see Methods and Additional file 1) with the annealing temperature set at either 70°C or 71°C as indicated. Amplified products were resolved using 8% PAGE and visualized by EtBr staining. The arrowheads point at bands amplified more prominently in the AR compared to the Control (IgG) lanes. M, marker DNA; numbers above bands indicate size in bps. B) Re-amplification and digestion. The two bands indicated in panel A by arrowheads were excised, purified and re-amplified with the same 'AC' and 'TG' primers used for CD. The products were subjected to secondary digestion with the indicated enzymes, followed by agarose gel electrophoresis. Arrowheads point at similar Hae III sub-fragments obtained from the two AR ChIPs. – C, no template control, UC, uncut, M, marker DNA. C) Mapping of AR target. The Hae III subfragments from B were excised, purified and sequenced. By blasting against the human genome using Ensembl , both sequences mapped to chromosome 17q25.3, ~1.5-kb upstream of the MAFG gene and ~2.5-kb downstream of the PYCR1 gene as shown in the diagram. The two genes are transcribed in the same direction as indicated by the horizontal arrows. pA, polyadenylation signal. The AR binding region discovered through CD (''hit'') abuts a CpG island (bottom, striped rectangle), but does not overlap with any repetitive elements (bottom, black rectangles). Several AREs (checkerboard triangles) were identified in this region using Consite . D) Validation of target by conventional ChIP analysis. AR occupancy at the PYCR1/MAFG locus was tested by conventional ChIP assay. The PSA enhancer serves as positive control. A non-target locus serves as the negative control. Genomic DNA was used to demonstrate that the ChIP amplification was performed within a dynamic range. – C, no template control. M, marker DNA.