Effects of BuGZ on AurA kinase activity in mitosis. (A–D) Cells were transfected with control, BuGZ, or TPX2 siRNA (si) or were treated with AurA inhibitor MLN8237 (100 nM) followed by staining with antibodies to total AurA (A) or p-AurA (B). Bars, 10 µm. The immunostaining intensity of total AurA (C) and p-AurA (D) in control siRNA–treated cells in metaphase or prometaphase (ProM, containing misaligned chromosomes or thick chromosome bars) and cells in the experimental groups containing misaligned chromosomes were quantified. 75–152 total cells from three independent experiments in each experiment were measured and quantified. Error bars indicate SEM. One-way ANOVA: *, P < 0.05; ***, P < 0.001. (E) Scheme of cell synchronization and mitotic shakeoff. (F) Cells transfected with siRNA were synchronized, collected, and analyzed by Western blotting with indicated antibodies. (G) XEEs were depleted (dep) with unimmunized rabbit IgG (IgG), xBuGZ, or xTPX2 antibodies or were treated with MLN8237. xAurA was immunoprecipitated and analyzed by Western blotting using indicated antibodies. (H) XEEs were depleted of xBuGZ and supplemented by purified YFP or YFP-xBuGZ followed by AurA immunoprecipitation and Western blotting. Noc, nocodazole; Thy, thymidine.