Extended Data Fig. 2: Purification of Kif3AB constructs, CRISPR knockout of Kif3B, and Kap3 imaging in cilia.

a, SDS-PAGE of purified Kif3AB constructs (post size-exclusion chromatography) with indicated C-terminal truncations. b, Genotype for homozygous Kif3B KO cell line, with insertions highlighted by alignment with the reference sequence. Clones were extensively Sanger sequenced to determine genotype (representative traces shown). c, Immunofluorescence images of cilia in Kif3B KO cell lines and cell lines stably expressing Kif3B^WT, Kif3B^ΔC156 and Kif3B^KKK. Cells were stained for gamma tubulin (yellow), acetylated tubulin (red) and DAPI (blue). d, Western blot showing expression of FLAG-tagged Kif3B^WT, Kif3B^ΔC156 or Kif3B^KKK in Kif3B KO cells, detected using anti-FLAG. GAPDH used as loading control. e. Top, representative images of mNeonGreen (mNG) tagged Kap3 in background of unlabelled Kif3B^WT and Kif3B^KKK constructs expressed in Kif3B KO cells. Bottom, plot of average mNG-Kap3 fluorescent signal from line scans along the cilium length (bars in top panel indicate distance analyzed). Kif3B^WT background, black. Kif3B^KKK background, green. Values are normalized relative to Kif3B^WT peak value. Traces show mean intensity ± s.e.m.; n = 39 (Kif3B^WT), 31 (Kif3B^KKK) cilia measured from three technical replicates.