Figure 2

A netrin-1-binding domain lies within the Aβ region of APP. (a) Schematic representation of APP. (b) HEK293T cells were cotransfected with a C100 (APP597-695) or C83 (APP613-695)-expressing construct in the presence or in the absence of a netrin-1 expression construct. Immunoprecipitation was performed using anti-APP (C-terminal) for the pull-down and an antibody raised against netrin-1 or against the C-terminal domain of APP for the immunoblot. (c) Netrin-1 immunoprecipitates with Aβ. In all, 150 ng of GST/netrin-1 were incubated with increasing concentrations of Aβ in 1ml of reaction buffer. Pull-down was performed using anti-Aβ-specific antibody, and netrin-1 was detected by western blot using anti-GST antibody. (d) An ELISA assay was performed as in Figure 1g; 0.18 µg/ml of Aβ or 0.07 µg/ml of Aβ1–17 protein was coated in 96-wells plate and various netrin-1 concentrations were added. Quantification of the interaction is indicated here by the measurement of the optic density (intensity)