Panel A shows immunocytochemical staining of Neuro 2 a cells transfected with s h R N A targeting K I F 3 A or C R I S P R-Cas 9 targeting K I F 3 B, with reduced immunoreactivity observed in transfected cells. Panel B quantifies the immunofluorescence intensity of K I F 3 A and K I F 3 B, showing significant signal reductions in knockdown and knockout cells. Panel C demonstrates sequential staining of Neuro 2 a cells expressing H A-K I F 3 A or myc-K I F 3 B with anti-H A and anti-K I F 3 A, or anti-myc and anti-K I F 3 B antibodies, showing punctate K I F 3 signals and colocalization. Panel D provides a schematic of K I F 3 A and K I F 3 B constructs and antibody epitopes. Panel E quantifies signal enrichment, showing no significant differences between staining orders. Panel F shows immunostaining of knockdown neurons, and Panel G quantifies K I F 3 A and K I F 3 B signal intensity in these neurons. Panel H shows immunostaining of C R I S P R-edited neurons, and Panel I quantifies K I F 3 B signal intensity in these neurons. Panel J shows immunostaining of rescue experiments. Panel K shows M A P 2 staining of neurons, confirming neuronal identity. Panel L quantifies the mean intensity per area of T R I M 46 and A n k G in the soma. Panel M provides a statistical summary. The table details the significance levels and mean differences between the various experimental groups tested. Panel N shows immunostaining to assess potential steric interference between antibodies at the axon initial segment (A I S), and Panels O, P, and Q quantify the minimum distances between different antibody pairs. All data is approximate.
Related to Figs. 1 and 2. (A) Immunocytochemical validation of anti-KIF3A and anti-KIF3B specificity. Neuro2a cells were transfected with an shRNA vector targeting KIF3A or a CRISPR-Cas9 vector targeting KIF3B and stained with the corresponding antibodies. EGFP and HA-Cas9 were used as transfection markers. Reduced immunoreactivity was observed in transfected cells. Arrows indicate KD or KO cells; arrowheads indicate non-transfected controls. Scale bar, 20 µm. (B) Quantification of KIF3A or KIF3B immunofluorescence intensity normalized to the mean of the corresponding control group (set to 1). Data are presented as bar graphs (mean ± SD) with individual data points overlaid. Significant signal reductions were observed in KIF3A KD and KIF3B KO cells (***, P = 2.54 × 10−4 and 2.60 × 10−14 by Welch’s t test). n = 17 and 21 cells (KIF3A control and KD); n = 17 and 18 cells (KIF3B control and KO). (C) Antigen detection of KIF3 antibodies. Neuro2a cells expressing HA-KIF3A or myc-KIF3B were stained with anti-HA and anti-KIF3A or anti-myc and anti-KIF3B antibodies. Sequential staining was performed in both orders. Punctate KIF3 signals and co-localization were observed under all conditions. Scale bar, 2 µm. (D) Schematic of KIF3A and KIF3B constructs and antibody epitopes. HA and myc tags were fused to the N termini of KIF3A and KIF3B, respectively. The anti-KIF3A monoclonal antibody recognizes a region spanning the distal stalk to proximal tail, whereas the anti-KIF3B polyclonal antibody was raised against full-length KIF3B. M, motor; S, stalk; T, tail. (E) Quantification of signal enrichment. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. Enrichment of the top 5% KIF3A or KIF3B signal within the top 5% HA or myc regions was quantified and normalized to randomly selected regions. No significant differences were detected between staining orders (n.s., P = 0.768214 and 0.502447 by Welch’s t test). n = 15 cells per condition. (F) Immunostaining of KD neurons. KD vectors were electroporated at DIV0, and neurons were immunostained with anti-KIF3A and anti-KIF3B antibodies at DIV4. Scale bar, 20 µm. (G) Quantification of KIF3A and KIF3B signal intensity in KD neurons. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. Signal intensities were normalized to the mean intensity of control neurons (set to 1). KIF3A: One-way ANOVA followed by Tukey’s post hoc test revealed a significant difference between KIF3A KD and KIF3B KD (***, P = 0.0002), a significant difference between KIF3A KD and control (***, P < 0.0001), and no significant difference between KIF3B KD and control (P = 0.4114). KIF3B: One-way ANOVA followed by Tukey’s post hoc test showed no significant difference between KIF3A KD and KIF3B KD (P = 0.9232), while significant differences were observed between KIF3A KD and control (***, P < 0.0001) and between KIF3B KD and control (***, P < 0.0001). n = 17 (control), 18 (KIF3A KD), and 19 (KIF3B KD). (H) Immunostaining of CRISPR-edited neurons. KO vectors were electroporated at DIV0, and neurons were immunostained with anti-KIF3B and anti-HA antibodies at DIV4. Scale bar, 20 µm. (I) Quantification of KIF3B signal intensity in gene-edited neurons. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. ***, P = 5.47 × 10−6 by Welch’s t test; n = 13 (control), 12 (KIF3B KO). (J) Immunostaining of rescue experiments. KO vectors together with KO-resistant myc-KIF3B were electroporated at DIV0, and neurons were immunostained with anti-myc and anti-HA antibodies at DIV4 to confirm double-positive cells. Scale bar, 20 µm. (K) MAP2 staining of the same neurons shown in Fig. 1 D, confirming neuronal identity and illustrating neurite orientation. (L) Quantification of mean intensity per area of TRIM46 and AnkG in the soma. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. No significant differences were detected by one-way ANOVA (P = 0.275 for TRIM46; P = 0.090 for AnkG). n = 12 (control), 12 (KIF3B KO), and 15 (rescue). (M) Statistical summary for Fig. 2 B. Each row represents a pairwise comparison between two groups. Group 1 and group 2 indicate the compared conditions, and meandiff represents the difference in mean values between group 1 and group 2. P-adj denotes the adjusted P value after correction for multiple comparisons. The lower and upper columns indicate the 95% confidence interval of the mean difference. The reject column indicates whether the null hypothesis was rejected (TRUE, statistically significant difference; FALSE, not significant). Tukey’s post hoc analysis showed that A > P and B > A were each significantly different from the remaining groups; however, the difference between A > P and B > A was not statistically significant. (N) Immunostaining to assess potential steric interference between antibodies at AIS. To test whether steric hindrance occurs during antibody binding, three staining conditions were performed for the following antibody pairs: KIF3A/KIF3B, KIF3A/KAP3, and KIF3B/KAP3: sequential staining in both orders and simultaneous staining. Arrowheads indicate representative puncta showing co-localized signals; arrows indicate representative puncta positive for KIF3B alone. Scale bar, 2 µm. (O) Quantification of the minimum distance from KIF3A to KIF3B and from KIF3B to KIF3A. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. No significant differences were detected among the three staining conditions (from KIF3A to KIF3B: P = 0.0799; from KIF3B to KIF3A: P = 0.0726 by one-way ANOVA). n = 18, 25, and 20 cells for the three staining conditions; 10 minimum-distance measurements were randomly sampled per cell. (P) Quantification of the minimum distance from KAP3 to KIF3A and from KIF3A to KAP3. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. No significant differences were detected among the three staining conditions (from KAP3 to KIF3A: P = 0.516; from KIF3A to KAP3: P = 0.0964 by one-way ANOVA). n = 25, 26, and 25 cells for the three staining conditions; 10 minimum-distance measurements were randomly sampled per cell. (Q) Quantification of the minimum distance from KAP3 to KIF3B and from KIF3B to KAP3. Data are presented as bar graphs (mean ± SD) with individual data points overlaid. No significant differences were detected among the three staining conditions (from KAP3 to KIF3B: P = 0.0852; from KIF3B to KAP3: P = 0.216 by one-way ANOVA). n = 29, 24, and 26 cells for the three staining conditions; 10 minimum-distance measurements were randomly sampled per cell. In O–Q, 3A and 3B denote KIF3A and KIF3B, respectively.