Human Tpr shares functional conservation with Drosophila Mtor. (A) Immunodetection of endogenous Tpr with a mouse mAb (red), ACA (green), and MTs (blue) in HeLa cells. (B and C) Luciferase (control) and Tpr RNAi cells were treated with nocodazole and processed for immunofluorescence with Mad2 (red) and ACA (green) antibodies. DNA (blue) was counterstained with DAPI. White boxed regions indicate the chromosome that is shown at a higher magnification on the right. (D) Quantification of Mad2/ACA pixel intensity at KTs for luciferase (median = 1.039, range = 0.44–4.45, n = 486 KTs/13 cells) and Tpr RNAi (median = 0.46, range = 0.09–1.69, n = 528 KTs/14 cells). The two populations are statistically different (P < 0.001; Mann-Whitney test). (E) Mitotic index in HeLa cells after luciferase and Tpr RNAi under physiological conditions or 16 h nocodazole treatment. Error bars represent SD from the mean obtained from three independent experiments. (F) IP from mitotic enriched parental HeLa cells (En) or HeLa cells stably expressing EGFP-Tpr (GFP). Load indicates total protein extracts. Purified beads (IP) were subjected to Western blot analysis for detection of interacting proteins. (G and H) IP from mitotic enriched HeLa extracts using an unspecific rabbit IgG (Un), rabbit anti-Mad2, or anti-Mad1 IgGs. (I) Colocalization of Mad1 (red) with Tpr (green) at nuclear pores but not at KTs (blue) during early prometaphase. MTs are shown in white. Inset shows a higher magnification of one KT pair without depicting MTs. (J) Proposed model for the role of Mtor/Tpr in the recruitment of Mad2 and Mps1 to unattached KTs after NEB. Ab, antibody; APC/C, anaphase-promoting complex/cyclosome; P, phosphorylation; Promet, prometaphase. Bars, 5 µm.