Fig. 3

Binding of cargos to ALS-associated KIF1A mutants. a, b Plasmids expressing Venus- or FLAG-tagged WT and missense-mutation KIF1A were transfected into HEK293T cells. Cells were lysed and immunoblotted with antibodies against EGFP (to recognize Venus) or FLAG. c, e HEK293T cells were cotransfected with plasmids encoding FLAG-RAB3A or FLAG-VAMP2, along with plasmids encoding Venus-KIF1A WT, designated mutants, or empty vector. FLAG-RAB3A or FLAG-VAMP2 was immunoprecipitated with a FLAG antibody, and the immunoprecipitated proteins were identified by blotting with the indicated antibodies. d Quantification of immunoprecipitates in c. The intensity of coimmunoprecipitated RAB3A was normalized to the intensity of RAB3A input, and then the value for each mutant was normalized to WT. n = 5 experiments. **P < 0.01. P = 0.0054 (WT vs R1100C); P = 0.0084 (WT vs R1356Q); P = 0.0570 (WT vs P1587L); P = 0.3055 (WT vs A1643V) by one-way ANOVA and Dunn’s test. Error bars represent SEM. f Quantification of immunoprecipitates in e. Quantification was performed as described in d. n = 5 experiments. *P < 0.05. P = 0.0281 (WT vs R1100C); P = 0.0192 (WT vs R1356Q); P = 0.0281 (WT vs P1587L); P = 0.0281 (WT vs A1643V) by one-way ANOVA and Dunn’s test. Error bars represent SEM