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. 2021 Jul 3;8(4):ENEURO.0066-21.2021. doi: 10.1523/ENEURO.0066-21.2021

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Copyright © 2021 Frei et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 3. — Cadherin-8 and cadherin-11 are expressed in synaptic compartments and cadherin-8 interacts with neuroligin-1. A, Forebrain tissues were subjected to synaptic fractionation analysis to determine the subcellular localization of cadherin-8 and cadherin-11. Markers (PSD-95 and syntaxin-1) were probed as control for purity of the fractionation. Total: total protein input, P1: nuclear, S1: cytosol/membranes, P2: crude synaptosome, S2: cytosol/light membranes, P3: synaptosome, SPM: synaptic plasma membrane, PSD: postsynaptic density, S3: synaptic vesicles. B, Quantification of protein enrichment in P3, SPM, PSD, and S3 fractions compared with total protein input. PSD-95: *p = 0.0159, ***p = 0.0002, ****p < 0.0001; Cdh8: p = 0.6618 (total vs SPM), p = 0.1673 (total vs PSD); Cdh11: *p = 0.0113, ****p < 0.0001; Syntaxin-1: ***p = 0.0009; one-way ANOVA with Dunnett’s multiple comparisons test. N = 8 P21 mice, two pooled forebrains per sample. Representative Western blotting of IP of (C) cadherin-8 and (D) cadherin-11 from P14 forebrain tissues. Cadherin-8 binds to cadherin-11, β-catenin, and neuroligin-1, whereas cadherin-11 binds to cadherin-8 and β-catenin, but not neuroligin-1. N = 3 independent co-IPs. Representative Western blotting of (E) IP of neuroligin-1-HA immunoblotted for Cdh8 and (F) IP of cadherin-8-myc immunoblotted for Nlgn1 from N2a cells overexpressing cadherin-8-myc and neuroligin-1-HA. Cadherin-8-myc is present in the IP of neuroligin-1-HA and neuroligin-1-HA is present in the IP of cadherin-8-myc. Cadherin-8 and neuroligin-1 are not expressed endogenously in N2a cells. FT, flow-through. N = 3 independent co-IPs. G, Immunocytochemistry of N2a cells overexpressing cadherin-8-myc (magenta) and neuroligin-1-HA (cyan), counterstained for DAPI (blue). Cadherin-8-myc and neuroligin-1-HA show partial co-localization at cell-cell contacts (arrowheads) and at the cell membrane (open arrowheads). Boxed areas are magnified. Scale bar: 20 μm.