SAC, spindle MTs, and Aurora B kinase activity in SENP6-depleted cells. (A–D) HeLa cells were treated with anti-SENP6 (−) or control (+) siRNAs and DTB for 48 h except in A, in which cells were treated with siRNA for 36 h and then with nocodazole (Noc.) for a further 12 h. Cells were fixed and stained for centromeres (CREST) and with antibodies against the indicated proteins, Mad2 (A) Bub1 (B), and α-tubulin (C and D). Bottom insets show Hoechst 33342 staining of the cells. (A and B) The boxed areas have been magnified and included as insets (top corners). Error bars represent the 95% confidence interval (***, P < 0.0001). (D) Cells were treated as in C but incubated at 0°C for 10 min before fixation. (E) HeLa cells were transfected with SENP6 or control siRNAs. 36 h after transfection, monastrol was added, and incubation was continued for 12 h. After monastrol wash out, the cells were allowed to recover in the presence or absence of ZM447439 (ZM) for 30 min, as indicated. The cells were lysed in sample buffer, and Western blots were performed with anti–phospho-MCAK (pMCAK; top) and α-tubulin (bottom) antibodies. (F) Phospho-MCAK signals from three separate experiments as shown in E were quantitated and normalized against the α-tubulin signal in the same samples. Mean normalized fluorescence intensity (NFI) signal is shown, and error bars indicate SEM. AU, arbitrary unit. Bars, 5 µm.