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.

Tpr is important for activating Mad1 and Mad2. (A) Immunofluorescence analysis. HeLa cells transfected with siRNA were arrested in mitosis by nocodazole for 6 h and stained with indicated antibodies. (B) The staining intensity of Mad1 at kinetochores (n = 300) was quantified from control or Tpr-depleted cells (n = 50, each) selected at random in prometaphase stage. (C,D) HeLa cells transfected with siRNA were arrested in mitosis by nocodazole followed by mitotic shake-off to allow collection. (C) Whole-cell extracts were prepared and resolved on sucrose density gradient by centrifugation. Gradients were separated into 16 fractions and precipitated with TCA. The indicated protein was identified with immunoblot analysis. (D) Immunoprecipitation analysis. (Lanes 3–10) Equal amounts of HeLa cell lysates were subjected to immunoprecipitation with anti-Cdc20 antibody, and bound Mad2 was determined by anti-Mad2 antibody. (E) HeLa cells transfected with siRNA were synchronized at the G₁/S boundary by thymidine double block. Cells were harvested at the indicated times after release from the G₁/S boundary and subjected to immunoblot analysis.

Tpr is important for mitotic checkpoint signaling. HeLa cells transfected with siRNA against Tpr or control nonsilencing siRNA. (A,B) FACS analysis. HeLa cells transfected with siRNA were treated with nocodazole for the indicated times and stained with anti-phospho-Histone H3 for the population of cells in mitosis and propidium iodide (PI) for the population of cells containing 4N DNA content. (C) Immunoblot analysis. (D) Confocal microscopy images. The cells were treated with nocodazole for 48 h and stained with anti-Tpr antibody and Hoeschst 33342. The graph summarizes the results of three independent experiments (n = 200–250); bars indicate standard deviations.

Tpr determines the NPC localization of Mad1 and Mad2. (A) Immunofluorescence analysis. HeLa cells transfected with siRNA were stained with indicated antibodies. The arrows indicate the interphase cells showing mislocalized Mad1 and Mad2 by depleting Tpr. The arrowheads indicate the Mad1-depleted interphase cells. (B,C) After siRNA transfection to HeLa cells, equal amount of total lysates were subjected to immunoprecipitation analysis using anti-Mad1 antibody, and the bound proteins were visualized with immunoblot analysis. (B) For lanes 9 and 10, HeLa cells transfected with siRNA were arrested in mitosis by nocodazole before immunoprecipitation analysis. (C) HeLa cells were separated to the nucleus (N) and the cytoplasm (C) prior to immunoprecipitation analysis. (Lanes 1–4) PARP1 and MEK1 were used as markers for the nuclear and the cytoplasmic fractions, respectively.

Tpr-depleted cells proceed to anaphase despite the presence of unaligned chromosomes. (A) Live cell time-lapse imaging. HeLa cells were transfected with a plasmid expressing GFP-H2B. Using nuclear envelope breakdown (NEBD) as T₀, the time of mitotic progression was measured from live cell movies. (B) Mitotic progression of 20 cells selected at random. Asterisk (*) indicates the cells containing errors in DNA segregation. (C) Immunoblot analysis. (D) Anaphase cells (n > 100) were scored as normal (blue), having misaligned chromosomes (purple), containing lagging chromosomes (yellow), and having failed to promote cytokinesis (light blue). The average from three independent experiments is shown with standard deviation. (E) HeLa cells were transfected with the indicated siRNA. Forty-eight hours after transfection, cells were incubated with MG132 (2 μM) for 3 h. Mitotic spindle and chromosomes were stained with anti-β-tubulin antibody and Hoechst 33258, respectively. The percentages of total metaphase cells (n > 100) displaying one to three (blue) or more than three (light blue) misaligned chromosomes (chromosomes located outside rectangular area possessing the central 30% of mitotic spindle), defined according to Meraldi and Sorger (2005), are shown.