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Fig. 1. A, characterization of antibodies to topoisomerase II{alpha} and ß by Western blotting of whole cell extracts from HeLa cells (left panel) and Ptk1 cells (right panel). Cells were dissolved in SDS sample buffer, electrophoresed on 5–20% polyacrylamide gels, and transferred to PVDF paper. They were then probed with antibodies to topoisomerase II{alpha}, with antibody to topoisomerase IIß or with a mixture of antibodies to topoisomerase II{alpha} and ß. The antibody to topoisomerase II recognizes a single band of Mr 170,000. The antibody to topoisomerase IIß recognizes a major band of Mr 180,000. In the HeLa extract, but not the Ptk1 extract, another band of Mr 150,000, thought to be a breakdown product of topoisomerase IIß, is also identified. In the final lane of the HeLa panel, preincubation of anti-topoisomerase IIß with an excess of the peptide used for immunization blocks the labeling of topoisomerase IIß. The peptide inhibited binding of anti-topoisomerase IIß to the Mr 180,000 band and the putative breakdown product. B, immunoprecipitation of topoisomerase IIß from mitotic cell extracts by anti-topoisomerase IIß peptide antibody. Mitotic cell extracts were precipitated with crude immune serum or with affinity-purified anti-topoisomerase IIß antibody linked to protein A beads. The beads were washed extensively, and attached proteins were dissolved in SDS sample buffer. Samples were run on 4–12% polyacrylamide gels, transferred to PVDF paper, and probed with anti-topoisomerase IIß antibody. One major specific band runs at Mr ~185,000 corresponding to the mobility of mitotic topoisomerase IIß. The dense band at Mr ~55,000 represents IgG heavy chain of the precipitating antibody. Mock immunoprecipitations in the absence of added cell extract reveal nonspecific background bands in the crude serum.