KIF4A turnover at the central spindle is regulated by PP2A–B56γ and -ε. (A) An EGFP-KIF4A HeLa cell line was treated with control or combined PP2A–B56γ and -ε siRNA for 36 h, arrested with 2 mM thymidine for 18 h, and then released for 8 h into fresh medium before imaging every 30 s as the cells passed through mitosis and cytokinesis. When cells in the field of view were in anaphase A, 1 µM ZM447439 was added to inhibit Aurora B and imaging, then continued during anaphase B. Arrows mark the central spindle region. (B) Central spindle staining intensity of EGFP-KIF4A in control and PP2A–B56γ and -ε silenced cells is plotted in the bar graph (mean ± SEM, n = 3). (C) Relative EGFP-KIF4A signal at the central spindle after Aurora B inhibition is plotted as a function of time on the line graph (means ± SEM, n = 3). (D) Turnover of EGFP-KIF4A was measured in control and PP2A–B56γ and -ε silenced cells using FRAP. In the example shown, the central spindle to be bleached is indicated by the dotted boxed areas, and arrows mark the recovering signal. (E and F) Turnover of the EGFP-KIF4A components at the central spindle (E) and chromosomes (F) are plotted in the line graphs (means ± SEM, n = 3).