Panel A shows a pull-down assay with G S T-tagged K I F 3 A and K I F 3 B dimers and their complexes with K A P 3, using mouse brain S 2 fraction. The results are evaluated by S D S-P A G E and Western blot, indicating the binding of various cargoes. Panel B presents four bar graphs quantifying the Western blot results, showing mean values with standard deviations. Panel C displays immunoprecipitation analysis using anti-K I F 3 B and anti-T R I M 46 antibodies, with quantitative analysis shown as a bar graph. Panel D includes Hi Res-S E C and dot blot analyses, probing the distribution of K I F 3 A, K I F 3 B, K A P 3, and T R I M 46, with results shown in a line chart and heatmap. Panel E quantifies protein distribution in different peak fractions, presented as bar graphs with mean values and standard deviations. The final proportions indicate that while K I F 3 A is restricted to the A B K fraction, T R I M 46 is significantly enriched in the B B K-associated fractions, defining two separate motor-cargo populations. All data is approximate.
KIF3/KAP3 subtypes exhibited cargo-binding specificity. (A) Pull-down assay assessing the binding capability of different KIF3/KAP3 complexes with reported cargoes. GST-AA, GST-BB, and GST-AB indicate GST-tagged homodimeric ACT (KIF3A 481–701), homodimeric BCT (KIF3B 475–747), and heterodimeric ACT/BCT, respectively. The S2 fraction of mouse brain lysate was used as input samples, and the results were evaluated by SDS-PAGE (top panel) and western blot (bottom panel). (B) Quantification analysis of the western blot results in A. Data are shown as bar graphs with mean ± SD. (C) IP analysis using anti-KIF3B and anti-TRIM46 antibodies with the brain S2 fraction as input. Rabbit normal IgG was also used as a control. Quantitative analysis was performed on the TRIM46 antibody IP group, in which the signal intensity of the IPed fraction was quantified as a percentage of its corresponding input based on densitometric measurements. Three replicates were performed, and data are shown as bar graphs with mean ± SD. (D) HiRes-SEC and dot blot analyses of the mouse brain S3 fraction probing the distribution of KIF3A, KIF3B, KAP3, and TRIM46. The elution fractions from SEC were used for dot blot analysis, with the results corresponding to their retention volumes in the SEC chromatogram (left panel). Quantification analysis was performed with five replicates and is shown as a line chart and heatmap (right panel). Elution positions of molecular weight markers are indicated: thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa). (E) Quantification of protein distribution. The top panel shows the proportion of KAP3 in the KIF3A/B/KAP3 (ABK, 23.5–26.5 ml) and KIF3B/B/KAP3 (BBK, 19.5–22.5 ml) peak fractions. The middle panel quantifies KIF3A and KIF3B protein levels partitioned into the ABK fraction. The bottom panel shows the Trim46 distribution in each KIF3 fraction. Data are presented as the percentage of each subunit recovered in the fractions relative to its respective total level. Bars represent mean ± SD from n = 3 independent experiments.