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Fig. 1. A, characterization of antibodies to topoisomerase II 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 520% polyacrylamide gels, and transferred to PVDF paper. They were then probed with antibodies to topoisomerase II, with antibody to topoisomerase IIß or with a mixture of antibodies to topoisomerase II 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 412% 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.