Fig. 6: Validation of SCRIB enrichment at the AIS in vitro and in vivo by immunostaining.

a Representative images of DIV14 neurons treated with or without 0.5% Triton X-100 extraction prior to fixation. Fixed hippocampal neurons were stained with SCRIB (red), AnkG (green, AIS), and MAP2 (blue, somatodendrites). Three independent experiments were performed. Scale bar, 50 µm. b Representative images of SCRIB knock-out using the CRISPR-Cas9 system. Hippocampal neurons were infected at DIV0 with AAV to express Scrib triple gRNA or template plasmids; both also harbor an HA expression cassette. Neurons were fixed at DIV14 and stained for SCRIB (magenta), AnkG (green), HA (red), and MAP2 (blue). Arrows indicate the AIS. Scale bar, 20 µm. c Quantification of the percentage of AIS SCRIB positive neurons. Seven independent experiments were performed using two different Scrib triple gRNAs to disrupt Scrib expression. The total number of quantified neurons in each condition was shown in the graph. n = 196, 184, and 192 neurons analyzed for control, Scrib gRNA1, and Scrib gRNA2, respectively. Data are mean ± SEM. One-way ANOVA, p = 6.97 × 10⁻¹¹. d Comparison of integrated AnkG intensity in the AIS 14 days after transduction with AAV to disrupt Scrib expression. Three independent experiments were performed. n = the number of neurons and is shown in the graph. Data are mean ± SEM. Unpaired two-tailed t test followed by Welch’s correction; ns, not significant. e Coronal cortical sections from P7 mice were stained for SCRIB (green), AnkG (red), and nuclei (DAPI, blue). Three independent experiments were performed. Scale bar, 50 µm.