Fig. 4.

Neo1 binds to Cbln4/Neurexin-1 complexes. (A) Schematic of cell aggregation assays in HEK293T cells to validate interaction of Neo1 and DCC with Cbln4. (B) Immunocytochemical validation of EmGFP (Emerald GFP), DsRed expression, and surface labeling of N1α−SS4⁺ and representative Neo1–SS1⁻;SS4⁻ (using anti-Flag antibody) and Cbln4 (using anti-HA antibody) in HEK293F cells. (C) Confocal images of cell aggregation assay showing a strong aggregation of green cells (coexpressing EmGFP, N1α-SS4⁺ and Cbln4) with red cell clusters (coexpressing DsRed and various Neo1 splice variants or Dcc). (D) Quantification of the aggregation index from the data in C and SI Appendix, Fig. S4. The binding of DCC and various Neo1 splice variants to Nrxn1α–SS4⁺/Cbln4 complexes was assessed, using pCMV (empty vector), Nrxn1β–SS4⁺ alone, and Nrxn1α–SS4⁺ alone as controls. Data are means ± SEM from two to three replicate experiments. Statistical significance was calculated by Tukey’s multiple comparisons test (two-way ANOVA). ****P < 0.0001, **P < 0.01, ns P > 0.05.