Fig. 1. Western blot analysis of cell extracts and supernatants of 293 cells expressing distinct ApoER2 variants. (A) Cartoon of the ApoER2 variants expressed in 293 cells and the recombinant soluble receptor fragment produced in E.coli. Numbered circles represent distinct ligand-binding repeats. F represents the domain carrying the furin cleavage site. A, B and C describe the cysteine-rich repeats of the EGF-homology domain. TM marks the transmembrane domain. MBP, maltose-binding protein; His, His₆ tag. (B) 293 cells were transfected with ApoER2Δ4-6,8-F (lane 1), ApoER2Δ4-6 (lane 2) or empty vector (lane 3). Ten micrograms of the corresponding cell extracts were separated on a 10% SDS–PAGE gel under reducing conditions and transferred to a nitrocellulose membrane. ApoER2 was detected by western blotting using an antibody recognizing the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit and a chemiluminescence system. (C) As (B), but western blotting was performed using an antibody against the cytoplasmic domain of the receptor (20). (D) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 4), ApoER2Δ4-6 (lane 2) or empty vector (lanes 3 and 5). The respective cell culture supernatants (1.5 ml) were incubated with 50 µl of RAP–Sepharose beads (lanes 1–3) or directly precipitated with TCA (lanes 4 and 5). The precipitated material was separated on a 15% SDS–PAGE gel under reducing (lanes 1–3) or non-reducing (lanes 4 and 5) conditions. For detection of the receptor fragment, Ab 220 was used in a western blot as described for (B). (E) 293 cells were transfected with ApoER2Δ4-6,8-F (lanes 1 and 2), ApoER2Δ4-6 (lanes 3 and 4) or empty vector (lanes 5 and 6) and incubated in the presence (+) or absence (–) of the furin inhibitor decanoyl-RVKR-chloromethylketone. The respective cell culture supernatants were used for detection of the soluble receptor fragment as described for (D).

Fig. 2. Recombinant ApoER2_1–3,7–MBP/His binds Reelin. (A) Five micrograms (lanes 1 and 2) or 0.5 µg (lanes 3–5) of recombinant ApoER2_1–3,7–MBP/His were separated on a 10% SDS–PAGE gel under reducing (lane 1) or non-reducing conditions (lanes 2–5). After transfer to a nitrocellulose membrane, lanes 1 and 2 were stained with Ponceau S, lanes 3 and 4 were processed for western blot analysis using the antibody against the first ligand-binding repeat of ApoER2 (220) (lane 3) or anti-MBP (lane 4), respectively. Lane 5 was incubated with Reelin-containing 293 medium and binding of Reelin was detected by western blotting using the antibody G10. Bound G10 was visualized with HRP–goat anti-mouse (1:15 000) and a chemiluminescence system. (B) Ten microliters of Reelin-conditioned medium were subjected to SDS–PAGE on a 6% gel, followed by a western blot using G10 as described in (A).

Fig. 3. (A and B) Binding of RAP-myc/His and Reelin to ApoER2_1–3,7–MBP/His. Ninety-six-well plates were coated with 10 µg/ml ApoER2_1–3,7–MBP/His. After incubation with the indicated amounts of either RAP-myc/His (A) or Reelin-containing 293T cell supernatant (B), bound RAP or Reelin was detected with anti-myc or anti-Reelin antibody and HRP-conjugated secondary antibody. (C) Ninety-six-well plates were coated with 10 µg/ml ApoER2_1–3,7–MBP/His (circles) or VLDLR_1–8–MBP/His (squares) and incubated with Reelin (1 nM) in the presence of increasing amounts of recombinant ApoER2_1–3,7–MBP/His (0–80 µg/ml). Bound Reelin was detected with anti-Reelin antibody and HRP-conjugated secondary antibody.

Fig. 4. Reelin-induced Dab1 phosphorylation is inhibited by the soluble ApoER2 fragment. Reelin-induced Dab1 phosphorylation was measured in primary mouse neurons after immunoprecipitation of Dab1 from cell lysates, followed by separation on an 8% SDS–PAGE gel and western blotting using anti-phosphotyrosine (PY) antibody and anti-Dab1 as described in Materials and methods. (A) Neurons were stimulated with control medium (lane 1), Reelin-conditioned medium (lane 2) or Reelin-conditioned medium in the presence of 40 µg/ml recombinant ApoER2_1–3,7–MBP/His (lane 3). Cells were processed for immunoprecipitation with anti-Dab1 and subsequent western blots were performed using anti-PY and anti-Dab1 as indicated. (B) Neurons were incubated with control medium (lane 1), Reelin-conditioned medium (lane 2), Reelin-conditioned medium in the presence of medium from 293 cells expressing ApoER2Δ4-6,8-F (lane 3) or in the presence of supernatant from 293 cells transfected with the empty plasmid (lane 4), medium from 293 cells expressing ApoER2Δ4-6,8-F (lane 5) or the empty plasmid (lane 6). Immunoprecipitation and western blot analysis as in (A).

Fig. 5. Analysis of splice variants of ApoER2 and detection of soluble ApoER2 in primary mouse neuronal cultures. (A) mRNA from embryonic brain from wild-type mice (lane 1), reeler mice (lane 3) and primary neuronal cultures (E15–16) (lane 2) was used for cDNA synthesis with reverse transcriptase and the resulting cDNA was used for PCR amplification as described in Materials and methods. Amplified products were separated on a 1.5% agarose gel. (B) Embryonic brains (E15–16) were dissected and the cerebrum (lane 1), the cerebellum (lane 2) and the olfactory bulbs (lane 3) were used for cDNA synthesis and PCR was performed as in (A). (C) Supernatant from a primary neuronal culture (E15–16) was immunoprecipitated with anti-Reelin antibody (G10). Immunocomplexes were precipitated with a mixture of protein A– and protein G–Sepharose and resolved on a 4.5–18% gradient polyacrylamide gel and transferred to nitrocellulose as described in Materials and methods. The soluble ApoER2 fragment was detected by western blotting using an antibody against the first ligand-binding repeat (220). Binding of the primary antibody was visualized with HRP–goat anti-rabbit (1:10 000) and a chemiluminescence system.

Fig. 6. Primary neurons in culture express and secrete Reelin. Primary mouse neuronal cultures (E15–16) were stained for the neuronal marker class III β-tubulin (TUJ1; red) and for Reelin (G10; green) as described in Materials and methods. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Roche). Bar: 30 µm. Inset: western blot analysis for the presence of Reelin in conditioned medium of primary mouse neurons. Supernatant (0.2 µl) from 293T cells expressing Reelin (lane A) and supernatant (15 µl) of a primary mouse E15–16 neuronal culture (lane B) were resolved on a 6% SDS–PAGE gel and tested for the presence of Reelin by western blotting using G10 as described in Materials and methods.