Figure 6

Hippocampal neurons respond to secreted Sema5B. (a) Supernatant from human embryonic kidney (HEK)293 cells overexpressing hemaglutinin (HA)-Sema5BΔC was immunoprecipitated using an HA antibody, separated by SDS-PAGE, and immunoblots probed with anti-HA antibody. N = 4. (b) Hippocampal neurons (2 days in vitro (DIV)) were cocultured with HEK293 cells transfected with HA-Sema5BΔC and immunolabeled with anti-HA (red) and anti-neurofilament (green) antibodies (anti-neurofilament antibodies also label HEK293 cells). Collapsed hippocampal growth cones (arrowheads) avoid HEK293 cells overexpressing HA-Sema5BΔC (yellow). In contrast, neurites extend along non-transfected HEK293 cells (arrows). (c, d) Hippocampal neurons (1 DIV) treated with supernatant from HEK293 cells expressing HA-Sema5BΔC exhibit collapsed growth cones ((d), arrowhead) compared to cells treated with untransfected HEK293 cell supernatant, which had typical growth cones that extended numerous filopodia ((c), arrows). N > 6 assays. (e) An example of a single collapse bioactivity assay. Collapse activity was determined at least twice for each aliquot of Sema5B-containing supernatant. Scale bar 20 μm.