Figure S1.

Identification of the minimal NEO1-NET1 interaction region, related to Figure 1

(A, B) SPR equilibrium binding experiments of different NET1 and NEO1 constructs. Graphs show a plot of the equilibrium binding response against used NEO1 construct concentrations (left panels: full-length NEO1 ectodomain (eNEO1), right panels: NEO1 FN type III domains 4 to 6 (NEO1_FN456). Ligands immobilized on SPR sensor chip: A, full-length NET1; B, NET1_ΔNTR. (C) Immunofluorescence staining of FLAG-tagged full-length human DCC (DCC_FL) and mouse NEO1 (NEO1_FL) overexpressed in COS-7 cells (green). Left panel: bound NET1_ΔNTR is stained via a Rho ID4 tag (red); right panel: transfected cells were incubated with buffer only as a negative control and stained as in the left panel. (D) Western blot of COS-7 cells transfected with the indicated plasmids used in C. α-tubulin serves as a loading control. (E, F) Proximity ligation assay (PLA) to test for simultaneous binding of NET1 and RGMB to NEO1. (E) COS-7 cells were transfected with a NEO1-mVenus fusion protein or the corresponding empty vector, and with full-length RGMB (wild type or RGMB-A186R). Transfected cells were incubated with NET1_ΔNTR before performing the PLA assay. PLA signals are shown in red and NEO1-mVenus transfected cells in green with nuclei in blue. (F) PLA signals were quantified and values from 3 individual experiments were plotted. A two-tailed, unpaired t test showed the statistical significance as p = 0.0107.