Fig. 3

IgSF21 binds and recruits neurexin2α for inhibitory presynaptic differentiation. a, b IgSF21-Fc binds specifically to COS-7 cells expressing extracellularly HA-tagged neurexin2α (HA-NRX2α) regardless of alternative splicing at site 4 (S4). For the binding assays, HA-NRX1αS1(+) S2(+) S3(+) S4(+/−), HA-NRX2αS1(+) S2(+) S3(+) S4(+/−) and HA-NRX3αS1(−) S2(−) S3(+) S4(+/−) variants were used. n = 30 cells for each construct from three independent experiments, Kruskal–Wallis one-way ANOVA, P < 0.0001. ***P < 0.0001 compared with HA-CD4 by Dunn’s multiple comparisons test. c Scatchard plot analysis shows that the Kd for the binding of IgSF21-Fc to HA-NRX2αS4(-) is 21.0 nM (n = 30 cells). d, e NRX2α-Fc binds to rat and mouse full-length IgSF21-HA (rIgSF21-HA, mIgSF21-HA), the mIgSF21-short isoform (mIgSF21S)-HA and mIgSF21-HA lacking Ig2 (∆Ig2), but not to mIgSF21-HA lacking Ig1 (∆Ig1). n = 30 cells for each construct from three independent experiments, Kruskal–Wallis one-way ANOVA, P < 0.0001. ***P < 0.0001 compared with HA-CD4 by Dunn’s multiple comparisons test. n.s., not significant. f, g Soluble NRX2α-Fc (15 nM) added to the coculture medium suppresses VGAT accumulation induced by mIgSF21-HA. n > 30 cells for each condition from three independent experiments, Kruskal–Wallis one-way ANOVA, P < 0.0001. ^# P = 0.0105 and ***P < 0.0001 by Dunn’s multiple comparisons test. h, i IgSF21-Fc-coated beads (filled arrowheads) induce NRX2α accumulation in contacting axons of cultured hippocampal neurons transfected with HA-NRX2α. An open arrowhead indicates an Fc-coated bead that physically contacts a transfected axon but has no significant accumulation of HA-NRX2α. Arrows indicate beads not contacting transfected axons. n = 75 beads for each condition from three independent experiments, Mann–Whitney test, ***P < 0.0001. Scale bars represent 30 μm a, d and 10 μm f, h. Data are presented as mean ± s.e.m