Fig. 4

USP35 maintains Aurora B stability by blocking APC^CDH1-mediated proteasomal degradation. a HEK293T cells were transfected with Myc-Aurora B alone or in combination with HA-CDH1, Flag-USP35, or Flag-USP35^C450A. The cell lysates were immunoblotted using the indicated antibodies. b HEK293T cells transfected with His-ubiquitin alone or in combination with Myc-Aurora B, HA-CDH1, Flag-USP35, or Flag-USP35^C450A were synchronized in prometaphase by treatment with 100 ng/mL NOC for 18 h and then treated with MG132 for 4 h. Aurora B ubiquitination was observed using a Ni-NTA-mediated pulldown assay. c HeLa cells transfected with CONi or USP35i were synchronized in prometaphase by a treatment with 100 ng/mL NOC for 18 h and then treated with MG132 for 6 h prior to harvesting. A western blot analysis was conducted to detect Aurora B protein levels. d HeLa cells transfected with CONi or USP35i were synchronized in prometaphase by a treatment with 100 ng/mL NOC for 18 h. The cells were then treated with 100 μg/mL cycloheximide (CHX) and harvested at the times indicated. A western blot analysis was conducted to detect Aurora B protein levels. Quantification of Aurora B levels was done considering the amount of β-actin protein in each case. e HeLa cells were transfected with Flag-USP35 or Flag-USP35^C450A and then treated with 100 μg/mL CHX. The cells were harvested at the times indicated. A western blot analysis was performed to detect Aurora B protein levels. Quantification of Aurora B levels was done considering the amount of β-actin protein in each case. The data in parts d and e from three independent experiments represent the mean ± SD (*P < 0.05, **P < 0.005, t-test)