Fig. 6: Cntn1 is an AIS protein. | Nature Communications

Fig. 6: Cntn1 is an AIS protein.

From: Antibody-directed extracellular proximity biotinylation reveals that Contactin-1 regulates axo-axonic innervation of axon initial segments

Fig. 6

a The knockout vector for in vitro transduction of neurons. b, c Immunostaining for Cntn1 (green), AnkG (red) and Map2 (blue) after transduction with control (b) or Cntn1 (c) sgRNAs; arrowheads indicate AIS. Neurons were transduced at DIV0 and fixed at DIV21. Scale bar, 10 µm. N = 8 independent experiments. d Quantification of Cntn1 knockout by sgRNA. N = 8 coverslips, 4 FOV for each coverslip and each sgRNA; n = 215 and 182 neurons for control and Cntn1 sgRNAs, respectively. P values calculated using a nonparametric two-sided T-test; no adjustments were made for multiple comparisons; (t = 14.07, df = 62) p = 5.79 × 10−21. e The Cntn1-myc vector used for infection of neurons. RO, retro-orbital. f Transduction of hippocampal neurons at DIV 10 using AAV to express myc-tagged Cntn1. Neurons were fixed at DIV14. Cntn1-myc (red) is enriched at the AIS (arrowhead) and colocalizes with β4 spectrin (green). Map2 (blue) labels somatodendritic domains. N = 3 independent experiments. Scale bar, 10 µm. g In vivo transduction of cortical neurons in 13-week-old mice to express myc-tagged Cntn1. Brains were collected 4 weeks later. Cntn1-myc (red) is enriched at the AIS (arrowhead) and colocalizes with β4 spectrin (green); Hoechst (blue) labels nuclei. N = 4 mice. Scale bar, 10 µm. h The percentage of transduced neurons with AIS Cntn1-myc. For in vitro experiments, N = 3, with 15 fields of view (FOV) and 86 transduced neurons. For in vivo experiments, N = 4, with 16 FOV and 72 transduced neurons. i The knock-in vector for in vivo tagging of Cntn1. DRS, donor recognition sites. AAV were delivered by intracerebroventricular (ICV) injection at P0 into Cas9 transgenic (Tg) mice. j In vivo transduction of P0 cortical neurons to tag endogenous Cntn1 (red). Brains were collected 8 weeks later. The smFP-V5 tagged Cntn1 colocalizes with AIS β4 spectrin (green, arrowhead). Hoechst dye (blue) labels nuclei. N = 4 mice. Scale bar, 10 µm. k The percentage of cortical neurons with AIS smFP-V5 labeling. N = 4 mice, with 15 FOV and 59 transduced neurons. In all violin plots dotted and dashed lines indicate quartiles and median, respectively. a, e, i Created with Biorender.com.

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