AβO binding is receptor-mediated but PrP^C-independent in Nanodiscs and mature neurons.

<p>Immobilized SMPL Nanodiscs were titrated with AβOs and bound AβOs were detected with NU2 oligomer-specific antibody coupled to an HRP-based colorimetric assay. Nonspecific binding was measured in the presence of excess ATA, which blocks AβO/receptor binding, and used to calculate the specific binding component (blue). n = 3; mean +/- SD. (a). To analyze Nanodisc proteins co-immunoprecipitating with AβOs, Nanodiscs containing biotinylated synaptic plasma membranes were affinity precipitated and visualized by SDS-PAGE immunoblot with biotin detection (b). To test the prediction that PrP^C mediates AβO binding, immobilized Nanodiscs were split into four equivalent reactions and pre-treated with 0, 0.05, 0.1, or 0.2 units of PIPLC to remove PrP^C before exposing to AβOs and probing with NU2 as in (a). PrP^C removal was verified by Western blotting after the colorimetric assay was complete. NU2 bound to each immobilized Nanodisc/AβO complex is detected in the blot by anti-mouse secondary antibodies used to probe for the mouse antibody against PrP^C (c). The effect of PrP^C removal on AβO binding was tested using mature hippocampal cultures treated with PIPLC (d). PrP^C was detected using an antibody (red) and fluorescence-conjugated AβOs were visualized directly (green).