Fig. 2. Recombinant Sema5A proteins inhibit RGC axons on a laminin substratum. (A) Domain structure of Sema5A, and diagrams of three Sema5A extracellular domain constructs. (B) Silver staining of the three Sema5A recombinant proteins (ECD-Fc, sema-Fc, TSP-Fc). (C) The percentage of growth cones on a laminin substratum exhibiting collapse in response to increasing amounts of oligomerized ECD-Fc. (D,E) Examples of growth cone responses to a substratum choice assay. (D) Retinal axons grew freely into regions containing heat-inactivated ECD-Fc (^*), but (E) avoided entering regions containing active ECD-Fc (^*). (F) The response rate of growth cones encountering a border of either heat-inactivated ECD-Fc or ECD-Fc. (G,H) Composite images showing the pattern and density of axon outgrowth for 25 explants grown on a laminin substratum in the presence of parental 293 cells (G), or Sema5A-myc expressing 293 cells (H), at 20% confluence. (I) Mean total neurite outgrowth per explant on a laminin substratum in the presence of HEK 293 cells transfected with full-length Sema5A-myc or parental cells. At both 20% and 40% confluence, explants grown in the presence of cells transfected with Sema5A-myc exhibited less neurite outgrowth. Scale bars: (D,E) 100 µm; (G,H) 250 µm.