Fig. 6. Netrin-1 binding to Unc5B mediates BBB integrity.
a, b Surface Plasmon Resonance measurements of anti-Unc5B-3 binding to human and rat Unc5B-ECD-Fc. c Dissociation constant for anti-Unc5B-3 binding to human and rat Unc5B. d Unc5B gene deletion strategy using tamoxifen injection. e Anti-Unc5B-3 was i.v. injected in P67 Unc5Bfl/fl or Unc5BiECko mice for 15 min. Mice were perfused and anti-Unc5B-3 binding was detected by immunofluorescence on brain sections using an anti-human IgG antibody, reproduced on n = 4 Unc5Bfl/fl and n = 3 Unc5BiECko brain. Western-blot (f) and quantification (g) of ECs treated with CTRL IgG or anti-Unc5B-3 for 1 h followed by recombinant mouse Netrin-1 treatment (500 ng/ml) for 10 min or 30 min. Each dot represents one independent experiment, n = 4 independent experiment. h Unc5B immunoprecipitation with a commercial antibody (R&D systems) of brain protein extracts from mice i.v injected with CTRL or anti-Unc5B-3 antibodies (1 h, 10 mg/kg), and western blot with antibodies recognizing the indicated ligands. i Quantification of h, n = 5 CTRL anti-Unc5B-1 and n = 5 anti-Unc5B-3 treated animals. Each dot represents one mouse. One control mouse was set as 1. j I.v. antibody injection strategy. k Immunofluorescence staining on brain sections from antibody-injected mice. l Quantification of brain cadaverine content, n = 5 CTRL IgG, n = 5 anti-Unc5B-3, n = 5 CTRL anti-Unc5B-1 and n = 4 anti-Unc5B-2 treated animals. Each dot represents one mouse. m I.v. antibody injection strategy. n Quantification of brain cadaverine content, n = 4 CTRL anti-Unc5B-1, n = 4 anti-Unc5B-3 and n = 3 anti-Unc5B-2 treated animals. Each dot represents one mouse. o I.v. antibody injection strategy. p, q Quantification of cadaverine content in peripheral organs, n = 4 CTRL anti-Unc5B-1, n = 5 anti-Unc5B-2, n = 5 CTRL IgG and n = 5 anti-Unc5B-3 treated animals. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.