Figure 4

Sumoylation-resistant Kif18A mutant induces a mitotic delay. (A) HeLa cells were co-transfected with a plasmid expressing His₆ -HA-WT or His₆ -HA-5R and a plasmid construct expressing His₆ -GFP for 36 h. Transfected cells were then treated with nocodazole (40 ng/mL) for 14 h, after which mitotic cells were collected by shake-off. Mitotic cells were then released into fresh medium. Cells were collected at various times of release and lysed in 8 M urea. Equal amounts of cell lysates were blotted for HA, cyclin B1, and GFP. Asterisk indicates phosphorylated form of His₆ -HA-Kif18A. Lane A denotes lysates from asynchronized cells to show the unmodified form of His₆ -HA-Kif18A. (B) HeLa cells transfected with GFP-WT or GFP-5R were subjected to time-lapse confocal microscopy analysis. The video-graphic process started when significant plus-end accumulation of GFP signals was observed. (C) Quantitative analysis of mitotic time of cells as shown in B. Data were summarized from three independent experiments (WT: n = 13; 5R: n = 13).