Fig. 7: AnkG is required for SCRIB enrichment at the AIS.

a, b Representative images of DIV3 (a) and DIV7 (b) hippocampal neurons stained for SCRIB (green), AnkG (red), and MAP2 (blue). Arrows indicate the proximal axon (a) and AIS (b). Scale bar, 20 µm. c Quantification of the percentage of neurons with AIS SCRIB and AnkG at DIV3, 7, 14, and 21. Four independent experiments were performed at each time point. Data are mean ± SEM. d Representative images of AnkG knock-out using the CRISPR-Cas9 system. DIV0 hippocampal neurons were transduced with Ank3 triple gRNA or template plasmids; the plasmids also harbor an HA expression cassette. Neurons were fixed at DIV14 and stained for SCRIB (magenta), AnkG (green), HA (red), and MAP2 (blue). Scale bar, 20 µm. e, f Quantification of the percentage of neurons with AIS AnkG (e) and SCRIB (f) after loss transduction with AAV to disrupt AnkG expression. Three independent experiments were performed with the number of neurons analyzed indicated on the figure. Data are mean ± SEM. Unpaired two-tailed t test, p = 0.0002 (e) and p = 0.0005 (f). g, h Co-immunoprecipitation of Scrib-Flag with AnkG270-EGFP. Scrib-Flag and AnkG270-EGFP or pEGFPN1 were co-transfected in HEK293T cells and immunoprecipitated by Flag antibody (g) or GFP antibody (h). Two independent experiments were performed. IP immunoprecipitation, IB immunoblotting, Rb-GFP rabbit anti-GFP, Rb-Flag rabbit anti-Flag, Ms-Flag mouse anti-Flag, Ck-GFP chicken anti-GFP. i Illustration of the full length and truncated SCRIB constructs. j Co-immunoprecipitation of truncated SCRIB with AnkG270. The experiment was performed once. IP immunoprecipitation, IB immunoblotting, Ck-GFP chicken anti-GFP, Ck-mCherry chicken anti-mCherry, Rb-GFP rabbit anti-GFP.