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Fig. 2. Cytological analyses of centromere-kinetochore proteins assembled on HAC and integration sites. (A,B) Metaphase chromosomes from different cell lines (S026 in A, and K031 in B and C) were analyzed by (a) FISH with an {alpha}21-I probe (green) and a BAC probe (red), or by (b-j and k-l) FISH with a BAC probe (red) in combination with immunofluorescence, using antibodies against CENPs (green). Chromosomes were counterstained with DAPI (blue). Arrows indicate the HAC or integration sites of the alphoid BAC. Arrowheads indicate the canonical centromeres on the host chromosomes. The typical variegated assemblies of CENPs at the integration site in BS-resistant K031 cells are shown in panel B as presence (assembly+) or absence (assembly–) of each CENP signal. (C) Small and large reformed minichromosomes (k and l, respectively) contain BAC integration signals (red) that overlap with CENP-A signals (green) in K031 cells under BS and G418 double-selective conditions. Bars, 10 µm.





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