MCAK suppresses centrosome separation and spindle length after monastrol washout by action at nonkinetochore microtubule tips. (A) A Western blot demonstrating depletion of endogenous MCAK protein with MCAK-specific siRNA and rescue with EGFP constructs in HeLa cell lysates. HeLa cells depleted of endogenous MCAK and rescued with EGFP (control), EGFP-IPNN, EGFP-wtMCAK, or EGFP–ΔN-term are shown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Intermediate expression of EGFP-IPNN and EGFP-wtMCAK suppresses spindle length compared with EGFP, but centrosomes still separate to form normal-length bipolar spindles. (C) High expression of EGFP-wtMCAK or EGFP–ΔN-term inhibits centrosome separation, whereas cells expressing EGFP or EGFP-IPNN form bipolar spindles. (B and C) Error bars are SEM. The data were thresholded to three expression levels from cell means of centrosome distances at four time points. There were no less than 75 cells measured for each construct at each time point. The total numbers of cells measured are 343 (GFP), 360 (IPNN), 401 (wtMCAK), and 339 (ΔN-term). (D) Images of metaphase cells expressing EGFP-wtMCAK or EGFP–ΔN-term. Note that EGFP–ΔN-term is absent from centromeres (arrow) but is present on microtubule tips (arrowheads) and at the centrosome. ACA and tubulin staining are shown. Bar, 10 µm.