Kif6 is a slow progressive motor. (A) Diagrams of kinesin constructs and summaries of their properties based on the results in this figure. rKin430 served as a positive control. N/D, not done. (B) Experimental scheme for protein expressions in HEK293T cells. Harvested cells were lysed for protein purifications using anti-Flag beads. (C) Coomassie blue-stained polyacrylamide gel showing a typical batch of purified proteins. (D) Schematic of invitro MT binding and single-molecule motility assays. Rhodamine-labeled, taxol-stabilized MTs were immobilized on Pluronic F127-treated glass surface of flow chambers via neutravidin-biotin interaction, followed by the addition of GFP-tagged kinesins at concentrations of 1–2 μM and ATP at 1–2 mM. Imaging was performed using TIRF microscopy. (E) Representative images of MT-binding assays. (F) Representative time-lapse images of single-molecule motility assays, cropped from Video 5. GFP-tagged full-length Kif6, Kif6-CC2, and rKin430 were added to immobilized MTs at 50, 5, and 1 nM, respectively. Typical GFP-positive puncta on MTs are tracked with arrows to show their behaviors over time. The corresponding kymographs are presented. (G) Velocities of indicated motor proteins on MTs. 180 puncta from three biological replicates were measured for each protein. Data are presented as mean ± SD plus sample dots. (H) Schematic of in vitro MT gliding assays. Motor proteins at concentrations of 2–4 μM were coated on the glass surface of flow chambers. After blocking the remaining non-specific binding sites with Pluronic F127, Rhodamine-labeled, taxol-stabilized MTs, and 1–2 mM ATP were added, followed by live imaging. (I) Representative image sequences of MT gliding assays, cropped from Video 6. Arrowheads denote the leading ends of representative gliding MTs, whereas asterisks indicate representative stationary MTs. (J) MT gliding velocities. 50 MTs from three biological replicates were measured for each protein. Data are presented as mean ± SD plus sample dots.