Figure 9.

Robo1 interacts directly with Nrp1. (A) E15.5 brain lysates were immunoprecipitated and immunoblotted with the indicated antibodies; note that Robo1 forms complexes with Robo2, Nrp1, Nrp2 and plexinA1, but not VEGFR3. (B) Cell lysates from COS-7 cells, transiently transfected with constructs for the indicated proteins or control expression vector, were immunoprecipitated with Fc-tagged Nrp1 or Robo1, and immunoblotted with anti-myc antibody. Robo1 bound homophilically to itself and heterophilically to Nrp1. (C) Covasphere aggregation assay to identify Robo1 heterophilic interactions. Beads coated with the indicated Fc proteins were imaged with a 25x objective at the end of the aggregation assay. Histograms show the corresponding changes in the proportion of beads (red, green and yellow) over time; heterophilic interactions are indicated by yellow. Robo1 appears to bind homophilically to itself, and heterophilically to Nrp1, but not Nrp2 or Sema3A. (D) Cell lysates from COS-7 cells, which were transiently transfected with myc-tagged full-length Nrp1, and incubated with Robo1-Fc, Robo1Δ1,2-Fc or Robo1Δ3,4,5-Fc in the culture medium, were immunoprecipitated with the same Fc-tagged protein and immunoblotted with anti-myc antibody. Robo1Δ1,2-Fc failed to co-immunprecipitate Nrp1, suggesting that the neuropilin binding domain resides within the first two Ig domains of Robo1. (E) Covasphere aggregation assays using Nrp-Fc (green) and Robo1-Fc, Robo1Δ1,2-Fc and Robo1Δ3,4,5-Fc (red) confirmed these findings.