Distribution and analysis of nucleosomal DNA associated with ectopic or total CENP-A immunoprecipitation. (A) Fluorescent in situ hybridization showing location of the DNA isolated from CENP-B and ectopic CENP-A immunoprecipitations. DNA isolated from either CENP-B (green) or ectopic CENP-A IP (red) was used to generate fluorescent in situ hybridization (FISH) probes and hybridized to metaphase chromosomes. Chromosome indicated by white arrow is shown magnified in the right panel. Blue/grey - DAPI, scale bars: 5 μm (left panel) and 2 μm (right panel). (B) High-resolution Bio-Analyzer DNA chips show no detectable mononucleosomal DNA in Mock immunoprecipitation (IP) and demonstrate that ectopic CENP-A nucleosome-associated DNA is octameric in size in normal colon, HeLa, and SW480 cells, ranging from 124 to 164 bp in length. (C) As above, indicating the size of nucleosome-associated DNA isolated from HeLa and SW480 cells by IP with anti-centromere antibodies (ACA) serum. (D) High-resolution Bio-Analyzer analysis of mononucleosomal DNA values for input, ectopic CENP-A nucleosomes (from Figure
1E) and centromere-specific CENP-A nucleosomes that were purified with ACA serum (from Panel E below). DNA lengths below 350 bp were binned into four categories, and plotted by percentage of total fluorescence (pg/uL). (E) Two-color WB analysis of CENP-A and H3 protein levels from HeLa and SW480 isolated by IP using ACA serum.