Fig. 1. CENP-C binds the alpha-satellite DNA in vivo. (A) HeLa cells were formaldehyde treated and chromatin was immunoprecipitated (Formaldehyde-ChIP) with the different sera. After reversal of crosslinking, the immunoprecipitated DNA samples were analyzed by dot blot. The level of enrichment of different human highly repetitive sequences is shown (left). Decreasing amounts of total DNA are included to provide an internal quantitation of the hybridization signals (right). The filters were sequentially hybridized with the following [-³²P]-labeled DNA probes: pZ7.6B (680 bp) and pZ21.A(850 bp) alpha-satellite DNAs, specific for chromosomes 7, 13 and 21; Sau3 A repeat (291 bp) and Alu repeat (300 bp). PI serum (rabbit preimmune serum), anti-CENP-B, anti-CENP-C and anti-ScII indicate the DNA recovered with the corresponding antibodies. (B) Densitometric quantitation of the hybridization signals. The intensity of the hybridization signal in the pre-immune samples was set to 1 arbitrary unit. (C) Level of enrichment of a single copy sequence (renin promoter region 350 bp) tested by semiquantitative PCR in the different immunoprecipitated samples. For each sample, three conditions of PCR (30-35-40 cycles) were used. Lanes 1-3, input (1 ng); lanes 4-6, PI serum; lanes 7-9, rabbit anti-CENP-B serum; lanes 10-12, rabbit anti-CENP-C serum. (D) HeLa cells were exposed to UV light for 15 minutes, lysed and chromatin was immunoprecipitated with the different sera. Immunoprecipitated DNA samples were hybridized with a cocktail of the pZ76.B and pZ21.A probes.