Figure 2

Kif18A is SUMO2-modified. (A) HeLa cells stably expressing His₆ -SUMO2 were co-transfected with Kif18A (or control) siRNAs and a plasmid construct expressing His₆ -GFP for 48 h. Transfected cells were then treated with 40 nM taxol for 16 h. At the end of treatment, cells were lysed in 8 M urea. Equal amounts of cell lysates were subjected to Ni-IDA pull-down analysis. Pull-down proteins were then blotted for Kif18A and GFP. Kif18A-S denotes SUMO2-modified Kif18A. (B) HeLa cells stably expressing His₆ -SUMO2 were transfected with a plasmid expressing FLAG-tagged SENP-1 or enzymatically defective SENP1 (SENP-1-Mut) for 48 h. 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 then blotted for Kif18A, Flag, and SUMO2. Notably, Flag-SENP1 was precipitated by Ni-IDA resin due to its histidine-rich property.