Figure 1.
Generation and basic characterization of Apoer2 EIG mice. A, Schematic representation of mouse Apoer2 demonstrating the cytoplasmic location of the wild-type NFDNPVY sequence and the mutated sequence EIGNPVY introduced into Apoer2 EIG. The position of the alternatively spliced exon 19 is indicated. The diagram is not drawn to scale. B, PCR genotyping of Apoer2 EIG homozygous (lanes 1 and 4), heterozygous (lane 2), and wild-type (lane 3) mice. C, The Dab1 protein interacts with wild-type Apoer2 but not the Apoer2 EIG mutant receptor. GST fusion proteins containing the cytoplasmic domains of wild-type Vldlr (lane 3) and Apoer2 (lane 5), but not the mutated Apoer2 EIG (lane 4) or GST control (lane 2), bound Dab1 from transfected human embryonic kidney 293 cells. D, Reelin signaling in Apoer2 EIG embryos is disrupted. Cultured primary neurons (E16; 5 d in culture) from Vldlr−/− (lanes 1 and 2), Apoer2−/−;Vldlr−/− (lanes 3 and 4), and Apoer2 EIG;Vldlr−/− (lanes 5 and 6) mice were treated with control (lanes 1, 3, and 5) or Reelin-conditioned (lanes 2, 4, and 6) medium. Lysates were collected, run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted using antibodies against the Apoer2 extracellular domain (α-ED) or C terminus (α-CT), phosphotyrosine to detect Dab1 phosphorylation (4G10), total Dab1, p-SFK, CDK5, serine phosphorylated Akt, total Akt, and serine phosphorylated GSK3β.