Figure 5. SUMOylation of NKAP is required for the kinetochore localization of CENP-E.

(a) Expression of siRNA-resistant RFP-NKAP (WT), but not RFP-NKAP (14KR) or RFP vector rescues chromosome misalignment caused by NKAP knockdown. Percentage of mitotic cells with misaligned chromosomes is shown. For quantifications, >100 mitotic cells were counted for each experiment and condition. Data are representative of three independent experiments and shown as mean±s.d. (b) Selected frames from time-lapse movies of representative cells as described in a. The time on the images is in minutes. Arrowheads indicate unaligned chromosomes. Scale bar, 10 μm. Full frames of all groups are shown in Supplementary Fig. 6a. See Supplementary Movies 4–9. (c) Complementation of Flag-NKAP (WT) but not Flag-NKAP (14KR) in NKAP-knockdown cells rescues CENP-E kinetochore localization. HeLa cells were transfected with indicated siRNAs and expressing plasmids for 72 h, followed by nocodazole treatment for 4 h. Cells were stained for CENP-E (green), CREST (red) and DNA (blue). The boxed kinetochore was enlarged to the bottom right of each merged images. Scale bar, 10 μm. (d) Quantification of relative fluorescence intensity of the CENP-E signal at kinetochores in cells as described in c (n>20 cells, >400 kinetochores for each group). Data are shown as mean±s.d. ***P<0.001 (one-way ANOVA). (e) Western blot analysis of siRNA-resistant Flag-NKAP (WT), Flag-NKAP (14KR) and endogenous NKAP expression in cells as described in c. Cells were randomly selected for observation and statistics.