Figure 7HeLa CCL2 cells treated with DMSO (control) or the Plk1 inhibitor, BI 2536 (1 μM) for 20 min were immunostained with a combination of either anti-total β-catenin and anti-Ect2 antibodies or anti-β-catenin p-S60 and anti-Ect2 antibodies (A). Scale bars, 10 μm. The signal intensities of total β-catenin, β-catenin p-S60, and Ect2 were measured in the midbody of compound-treated telophase cells (B). A dot density graph with a box plot shows the signal intensity of midbody-localized β-catenin, β-catenin p-S60 or 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. More than 100 cells from three independent experiments were measured. ***, p<0.001; NS, not statistically significant (unpaired two-tailed t test); Scale bars, 10 μm. (C) After 18 h of nocodazole (200 ng/mL)-induced cell cycle arrest, HeLa CCL2 cells transfected with HA-Ect2 were released by incubation in fresh medium for 2 h and treated with either DMSO (control) or Plk1 inhibitor (BI 2536) for the indicated times. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The band intensity of β-catenin bound to HA-Ect2 was quantified with ImageJ and normalized to the signal intensity of immunoprecipitated HA-Ect2, and the relative values are shown below the bands. (D) HeLa CCL2 cells transfected with HA-Ect2 were infected with sh-control luciferase (shGL) or shPlk1 lentiviruses. The resulting cells were used for immunoprecipitation analysis using anti-HA antibody, and immunoprecipitates were separated by 10% SDS-PAGE followed by immunoblot analysis with the indicated antibodies. (E) HeLa CCL2 cells were co-transfected with GFP−β-catenin (+GFP-β-catenin) and either empty vector (+vector, control), HA-Plk1 kinase-dead mutant (+KM), or HA-Plk1 constitutively active mutant (+T210D), then treated with 200 ng/mL nocodazole for 18 h and released by incubation in fresh medium for 2 h (telophase). The resulting cells were used for immunoprecipitation analysis with anti-Ect2 antibodies. Immunoprecipitated beads were separated by 10% SDS-PAGE followed by immunoblot analysis using the indicated antibodies. (F) HeLa CCL2 cells were co-transfected with HA-Ect2 and either wild type Flag-β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D) and then treated with 200 ng/mL nocodazole for 18 h and released by incubation in fresh medium for 2 h (telophase). The resulting cell lysates were used for immunoprecipitation analysis with anti-HA antibody. Immunoprecipitated beads were separated by 10 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. (G) HeLa CCL2 cells were transfected with either wild type Flag−β-catenin (+WT), Flag-β-catenin S60A mutant (+S60A), or Flag-β-catenin S60D mutant (+S60D), then treated with 200 ng/mL nocodazole for 18 h (metaphase) and released by incubation in fresh medium for 2 h (telophase). Cell lysates were used for pull-down analysis with rhotekin-RBD beads, and the pulled down beads were separated by 12 % SDS-PAGE followed by immunoblot analysis with the indicated antibodies. The membrane was then stained with Coomassie (CBB) to determine the amount of RBD precipitated.