Figure 4.

Kif3a is required to localize p150^Glued to the subdistal appendage. (A) WT and Kif3a^−/− total cell lysates were subjected to immunoprecipitation of Kif3a, p150^Glued and c-Myc, which served as a negative control. Precipitating proteins were subjected to immunoblotting for Kif3a and p150^Glued. (B) Schematic representations of the motor domain and the truncation mutants of GFP-tagged Kif3a. (C) We expressed the indicated GFP-tagged versions of Kif3a in HeLa cells and immunoprecipitated GFP. Endogenous p150^Glued and each of the Kif3a fusion proteins were detected by immunoblotting for p150^Glued and GFP, respectively. (D) Expression of the full-length GFP-tagged Kif3a and the truncated mutant that interacted with p150^Glued (‘g’, green) restored the localization of p150^Glued (‘p’, red) to the subdistal appendage in Kif3a^−/− cells. (E) Quantification of p150^Glued localization to the subdistal appendage. (F) Expression of the motor dead GFP-tagged Kif3a (K106A and T107N) mutants ('g', green) restored the localization of p150^Glued (‘p’, red) to the subdistal appendage of Kif3a^−/− MEFs. (G) Quantification of p150^Glued localization to the subdistal appendage in Kif3a mutants. (H) We immunoprecipitated the GFP tag of the motor dead Kif3a forms and detected endogenous p150^Glued and GFP. At least 100 cells were analysed per experiment (n=3), P<0.001 (paired t-test). Scale bars indicate 5 μm for all images.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 4C and 4H ^{(1.1MB, pdf)}