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Fig. 2. Mitotic chromosomes isolated from HeLa cells contain both topoisomerase II{alpha} and ß. A, isolated chromosomes were analyzed by Western blotting with antibody to topoisomerase II{alpha}, antibody to topoisomerase IIß, antibody to topoisomerase IIß preincubated with blocking peptide, antibody to topoisomerase IIß preincubated with nonspecific peptide, and a mixture of antibodies to topoisomerase II{alpha} and ß. B, fractions prepared during the isolation of mitotic chromosomes were analyzed by Western blotting for the presence of topoisomerase II{alpha} and ß. The lanes show the final isolated chromosomes, the cytoplasm remaining in the supernatant after pelleting of the chromosomes, the low-speed pellet containing large pieces of cellular debris and clumps of interphase nuclei, and nuclei containing contaminating interphase nuclei and aggregates of mitotic chromosomes. Topoisomerase II{alpha} was present in all lanes that contain either chromosomes or interphase nuclei but was not detected in the cytoplasm. Topoisomerase IIß was present at high concentration in all lanes that contain either chromosomes or interphase nuclei. A small amount of topoisomerase IIß was detected in the cytoplasmic extract, suggesting that either some topoisomerase IIß was not associated with the chromatin or that it was released during cell fractionation. C, isolated mitotic chromosome was triple labeled to reveal topoisomerase II{alpha}, topoisomerase IIß, and the distribution of DNA. Antibodies to both topoisomerase II isoforms label along the chromosome arms.