Figure 3
EphB2 interacts with the amino-terminal domain of Reelin. (A) Schematic diagram of partial Reelin constructs. The dark- and bright-green rectangles indicate the signal peptide, the F-spondin homology region and the unique region that is recognized by the G10 anti-Reelin monoclonal antibody (arrowhead). The circles with numbers represent the eight Reelin repeats. The carboxy-terminal myc-tags of the partial Reelin constructs are highlighted in orange. Arrows indicate sites of proteolytic Reelin cleavage. (B) The Reelin constructs shown in A were transfected into HEK-293 cells. Supernatants were separated by SDS-PAGE and proteins were detected with an anti-myc antibody. Comparable amounts of the constructs were used for the coprecipitation experiments shown in C and D. (C) Fc-tagged ApoER2 or (D) EphB2 ectodomains were incubated with supernatants containing similar amounts of the different myc-tagged partial Reelin polypeptides and precipitated with protein A/G-sepharose. Coprecipitated Reelin constructs were visualized by immunoblotting with an anti-myc antibody. The central fragment containing Reelin repeats R3-6 binds to the lipoprotein receptor ectodomains (C), whereas EphB2 binds to the amino-terminal Reelin fragment N-R2 (D).
