Figure 4

PlexinA1 inhibits in vivo and in vitro Fps and Fer autophosphorylation. (A), HEK293T cells were co-transfected with expression plasmids encoding Myc epitope-tagged Fps or Fer with or without PlexA1 as indicated. Fps or Fer were immunoprecipitated with anti-Myc antibody and immunoblotting with anti-pY was used to assess their degree of in vivo tyrosine phosphorylation (upper). The blot was then stripped and reprobed with anti-Myc to assess the level of Myc-tagged Fps and Fer expression (lower). (B), Fps and Fer were immunoprecipitated from neonatal mouse brains from animals of the indicated genotypes. Immune complex kinase assays were then performed with (+) or without (-) addition of purified GST-PlexA1-cyto (cytoplasmic domain) fusion protein as an exogenous substrate. Immunoblotting with anti-pY revealed the degree of Fps or Fer autophosphorylation (upper). The blot was then stripped and reprobed with anti-Fps/Fer (lower).