Panel A shows immunostaining of T R I M 46 in knockdown neurons, with arrows indicating T R I M 46 accumulation above a defined intensity threshold. Panel B presents a statistical analysis of axon initial segment (A I S) length in knockdown experiments, shown as bar graphs with mean and standard deviation (S D) and dot plots. Panel C shows the percentage of neurons with A I S length less than 5 micrometers. Panel D displays immunostaining of Ankyrin G (A n k G) and T R I M 46 in C R I S P R-mediated knockout neurons expressing H A-Cas 9, with arrows indicating A n k G and T R I M 46 accumulation above a defined intensity threshold. Panel E provides a statistical analysis of A I S length in knockout experiments, shown as bar graphs with mean and S D and dot plots. Panel F shows western blotting results of cycloheximide (C H X) chase experiments. Panel G presents western blotting results of K I F 3 B ubiquitination, with myc-K I F 3 B-His 7 purified under denaturing conditions in the presence of 6 M guanidine. Panel H shows a line graph of the results of C H X chase experiments, with protein levels of myc-K I F 3 B-His 7 normalized to the level at 0 hours after C H X treatment. Panel I quantifies ubiquitination, shown as bar graphs with mean and S D and dot plots. The graphs collectively illustrate the impact of K I F 3 A and K I F 3 B knockdown on T R I M 46 accumulation and A I S length in neurons. All data is approximate.
KIF3B is required for TRIM46 accumulation at the AIS. (A) Immunostaining of TRIM46 in KD neurons. Arrows indicate TRIM46 accumulation above a defined intensity threshold. Scale bar, 20 µm. (B) Statistical analysis of AIS length in KD experiments. Data are shown as bar graphs with mean ± SD and dot plots. One-way ANOVA followed by Tukey’s post hoc test. *, P = 0.0417 (control vs KIF3A KD) and ***, P = 0.0003 (control vs KIF3B KD). n = 30 (control), 33 (KIF3A KD), and 49 (KIF3B KD). (C) Percentage of neurons with AIS length <5 µm. (D) Immunostaining of AnkG and TRIM46 in CRISPR-mediated KO neurons expressing HA-Cas9. Arrows indicate AnkG and TRIM46 accumulation above a defined intensity threshold. Scale bar, 20 µm. (E) Statistical analysis of AIS length in KO experiments. Data are shown as bar graphs with mean ± SD and dot plots. One-way ANOVA followed by Tukey’s post hoc test revealed a significant difference between control and KIF3B KO (**, P = 0.0058), a significant difference between KIF3B KO and rescue (*, P = 0.013), and no significant difference between control and rescue (P = 0.8762). n = 12 (control), 12 (KIF3B KO), and 15 (rescue). (F) Western blotting of CHX chase experiments. (G) Western blotting of KIF3B ubiquitination. Myc-KIF3B-His7 was purified under denaturing conditions in the presence of 6 M guanidine, and ubiquitination was assessed; no marked ubiquitination was detected. (H) Line graph showing the results of CHX chase experiments. Protein levels of myc-KIF3B-His7 were normalized to the level at 0 h after CHX treatment (set to 1.00). At each time point, no significant difference was detected between co-transfection with full-length TRIM46 and TRIM46 ΔRING. Data are shown as line plots representing the mean ± SD, together with individual data points. P = 0.958, 0.986, 0.466, 0.224, and 0.772 for 0.5, 1, 2, 4, and 6 h, respectively, by Welch’s t test. n = 5 biological replicates. (I) Quantification of ubiquitination. Data are shown as bar graphs with mean ± SD and dot plots. Signal intensities of ubiquitin immunoreactivity above 100 kDa were measured and normalized to myc-KIF3B-His7. No significant difference was detected between co-transfection with full-length TRIM46 and TRIM46 ΔRING. P = 0.653 by Welch’s t test. n = 4 biological replicates. Source data are available for this figure: SourceData F1.