NEKL-3 phosphorylates OSM-3 in vitro. (A) SDS-PAGE result of the purified NEKL-3 proteins. (B) Western blot illustrating phosphorylation of OSM-3 by NEKL-3. (C) Relative ATPase activity of phosphomimic OSM-3^S96D normalized to kinesin heavy chain (KHC). The results were obtained from three independent experiments. Statistical significance, compared with OSM-3^WT with a matching color code, is based on Student’s t test. ***P < 0.001. (D) Schematic of single-molecule motility assay for the measurement of motility properties of OSM-3 phosphorylated by NEKL-3. (E) Representative kymographs showing the non-active motion of OSM-3^S96D along the MT. Horizontal scale bar, 5 µm; vertical bar, 10 s. (F) Alignment of motor domain sequence between human KIF17 and C. elegans OSM-3 proteins. Identical residues are highlighted in purple. R10 and S96 of OSM-3 are highlighted in scarlet. Source data are available for this figure: SourceData FS3.