CDP binding sites in HS3. (A) FP19, FP20, FP16, and FP1 contain binding sites for CDP. In this competition scan, the FP19 probe was labeled and binding assays were performed with K562 extracts. Incubations were performed at 4°C and gels (3.5%) were also run at 4°C for 3.5 hours to enable detection of the CDP complex, which is large and has a tendency to degrade. Competitors (100 ng) were added as indicated above the lane. Lane 1 has no competitor (0). The final lane (CDP) confirms that the slowest complex is efficiently competed by an oligonucleotide containing a well-characterized CDP binding site.46 (B) Anti-CDP antibody inhibits formation of the CDP complex. In the left panel, the CDP oligonucleotide (Table 1) was used as a probe; in the right panel, the probe was FP19. For both panels, lane 1 consists of a standard binding assay (no antibody preincubation); in lane 2, 0.5 μL of anti-CDP antibody was preincubated with extract protein before probe addition as described in Materials and Methods; in lane 3, a similar volume of preimmune serum was added in the preincubation step. Formation of the CDP complex (arrowhead) is abolished in the presence of antibody.