Figure 3

Identification of Kif18A sumoylation sites. (A) Schematic representation of lysine residues of wild-type human Kif18A. Key lysine residues subjected to mutational analsyis are indicated. HA and His₆ tags are fused in-frame at the C-terminus. Three major functional domains of Kif18A are also shown. (B) HeLa cells were transfected with plasmids expressing His₆ -HA-tagged Kif18A (His₆ -HA-WT) or the mutant protein with 5 lysine residues (K148, K442, K533, K660 and K683) replaced with arginines (His₆ -HA-5R) for 48 h. Flag-SUMO2 was also used for co-transfection. Transfected cells were then treated with 40 nM taxol for 16 h. At the end of treatment, cell pellets were lysed in 8 M urea. Equal amounts of cell lysates were subjected to Ni-IDA pull-down analysis. Pull-down proteins were blotted for HA and Flag signals. Flag blots of both short and long exposure are shown.