Figure 3.

NGF reverses neurofascin-dependent cell segregation and inhibits neurofascin-dependent cell aggregation. Neuroblastoma cells expressing either native neurofascin or the FIGQY→F mutant of neurofascin were incubated with NGF and evaluated for tyrosine phosphorylation of neurofascin (A), ability to direct cell segregation (B and C), and cell aggregation activity (E). (A) HA epitope-tagged neurofascin or FIGQY→F mutant neurofascin were immunoprecipitated from extracts of cells incubated 30 min in the presence or absence of NGF (100 ng/ml). Immunoprecipitates were resolved by SDS/PAGE, and analyzed for phosphotyrosine immunoreactivity by immunoblotting with an antiphosphotyrosine polyclonal antibody (10). (B and C) Unlabeled cells expressing either native neurofascin (B) or FIGQY→F-mutated neurofascin (C) were mixed in a 1:1 ratio with DiA-labeled untransfected cells and incubated for 30 min resulting in aggregates as in Fig. 1. Cells were then further incubated in the absence or presence of NGF (100 ng/ml) for 30 min. (D) Measurement of cell aggregation as a function of time in the presence and absence of NGF (100 ng/ml) for cells expressing native or the FIGQY→F mutant neurofascin (Fig. 2E).