Figure 4

Reelin induces EphB2 tyrosine phosphorylation in cortical neurons. (A) Primary neurons were treated with recombinant Reelin or preclustered Fc-ephrin B1. Before immunoprecipitation with an anti-EphB2 antibody, the cell lysates were analyzed by western blotting with the same antibody (IB: EphB2; input). The immunoprecipitated samples (IP: EphB2) were analyzed by western blotting with a phosphotyrosine-specific monoclonal antibody (IB: pTyr) to determine tyrosine phosphorylation levels of the EphB2 receptor in Reelin- or Fc-ephrin B1-treated neurons compared to control treatment. (B) Primary neurons were preincubated with GST or GST-RAP before stimulation with Reelin or preclustered soluble ephrin B1. The lipoprotein receptor antagonist RAP prevented the Reelin-induced tyrosine phosphorylation of Dab1 as detected by immunoblotting with the phosphotyrosine-specific antibody (middle blot, lane 5), but did not block the induction of EphB2 tyrosine phosphorylation (bottom). Shown are representative blots of at least three independent experiments (A-B). (C) Primary neurons lacking the adapter protein Dab1 (Dab1^−/−) were treated with Reelin or preclustered Fc-ephrin B1 (n = 2 knockout embryos). Deficiency in the Src family kinase switch protein Dab1 did not prevent Reelin- or ephrin B1-induced EphB2 phosphorylation. (D) Primary neurons deficient in VLDLR or both Reelin receptors ApoER2 and VLDLR were treated with Reelin or preclustered soluble ephrin B1. Lack of VLDLR (VR^−/−) or VLDLR and ApoER2 (VR^−/−;ER2^−/−) did not block the induction of EphB2 tyrosine phosphorylation by Reelin or ephrin B1. Neurons were prepared from three different double knockout embryos.