Tpr binds to Mad1 and Mad2 directly. (A,D) Total cell lysates were prepared from HeLa cells grown asynchronously or arrested in mitosis by nocodazole followed by mitotic shake-off to allow collection, and then subjected to immunoprecipitation analysis. (B,E) The indicated proteins were expressed in HEK293 cells and subjected to GST pull-down analysis. (C, lanes 1,2) Coomassie staining of purified recombinant GST-tagged proteins. (Lane 3–5) Autoradiography of in vitro translated Tpr proteins. (Lanes 6–11) GST pull-down analysis was performed, and the bound proteins were visualized by autoradiography. (F, lanes 1–4) Coomassie staining of purified recombinant GST-tagged proteins. (Lanes 5–8) GST pull-down analysis was performed, and the bound proteins were visualized by autoradiography.