Figure 6(A) Asynchronously growing HeLa CCL2 cells were immunostained with the indicated antibodies after extraction using 0.2% Triton X-100. The resulting samples were observed using a confocal laser microscope (Zeiss LSM 700). Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (A). Arrowheads indicate Ect2, total β-catenin, and β-catenin p-S60 located at the midbody in telophase HeLa CCL2 cells. (B) Intercentrosomal (centrosome-to-centrosome) linear signal profiles of β-catenin/Ect2 (upper panel) and β-catenin p-S60/Ect2 (lower panel) from the XY plane of the immunostained cells in (A). All immunofluorescence images and linear signal profiles show representative data of more than 50 telophase cells obtained from three independent experiments (A, B). (C) Asynchronously growing HeLa CCL2 cells were transfected with HA-Ect2. The resulting cells were used for immunoprecipitation analysis using either anti-HA antibody or non-immunized mouse IgG control. Precipitates were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies. (D) HeLa CCL2 cells co-transfected with HA-Ect2 and GFP-β-catenin were synchronized using a double thymidine block and released using medium containing 200 ng/mL nocodazole (Noc.) at the indicated time point. Cells at each release time point were harvested and used for immunoblot analysis using anti-HA antibodies. Immunoprecipitated beads were analyzed by SDS-PAGE followed by immunoblot analysis. Band intensities were quantified using the ImageJ program and normalized to the level of immunoprecipitated HA-Ect2 protein, and the relative values of band intensities are marked. (E, F) HeLa CCL2 cells infected with the indicated lentiviruses were co-transfected with HA-Ect2 and Flag-Plk1. Cells were then treated with nocodazole (200 ng/mL) for 18 h and released from cell cycle arrest by incubation in fresh medium for 6 h. The resulting cells were used for immunoprecipitation analysis using anti-Flag (E) or anti-HA (F) antibodies. Precipitated beads were used for immunoblot analysis using the indicated antibodies. (G, H) HeLa CCl2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were co-immunostained with anti-β-catenin and anti-Ect2 antibodies. Localization of β-catenin and Ect2 in the midbody of telophase cells was observed using a confocal microscope (G) and the signal intensity of Ect2 was monitored in the midbody of each knockdown telophase cell (H). Arrows indicate total β-catenin and Ect2 located at the midbody in telophase HeLa CCL2 cells (G). A dot density graph with a box plot shows the signal intensity of midbody-localized Ect2 in telophase cells. The box plot represents: central blue band, Median; box, Interquartile range; upper whisker, Maximum observation below upper fence; lower whisker, Minimum observation above lower fence; dot, Outlier. Approximately 70 cells from three independent experiments were measured. ***, p<0.001 (unpaired two-tailed t test); Scale bars, 10 μm. The value of the relative neck thickness is shown in the lower left corner of the merged images (G). (I) HeLa CCL2 cells infected with either sh-control luciferase (shGL) or shβ-catenin lentiviruses were treated with nocodazole (200 ng/mL) for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were analyzed by pull-down assays using rhotekin RBD beads, and pulled-down proteins were separated by SDS-PAGE followed by immunoblot analysis using the indicated antibodies. The membrane was then stained with coomassie (CBB) to confirm the amount of RBD precipitated.