Confirmation of the identity of some CC-3 antigens by immunoblotting. (A) Mitotic cell extracts were immunoprecipitated with either CC-3 and MPM-2 and immunoblotted with different antibodies against proteins suspected to be CC-3 antigens. Lysates from asynchronous (AH) and mitotic (MH) HeLa cells were also electrophoresed and immunoblotted. Both CC-3 and MPM-2 immunoprecipitates contained hyperphosphorylated forms of Rpb1 and hSpt5, the α and β isoforms of DNA topoisomerase II and p54nrb/nmt55. However, only CC-3 is able to precipitate mitotic SAP155. (B) Mitotic cell extracts were immunoprecipitated with antibody NMT-5, a rabbit polyclonal raised against p54nrb/nmt55 and immunoblotted with MPM-2, CC-3 and NMT-1, a monoclonal to p54nrb/nmt55. Note that a still unidentified mitotic antigen migrating more slowly than p54nrb/nmt55 in the mitotic HeLa cell sample is reactive with CC-3 in this region of the gel (asterisk). IP: immunoprecipitating mAb, IB: immunoblotting mAb.