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.
