Figure 3. APP ectodomain binds to NgR1-expressing cells. a, A schematic illustrates the cellular orientation of APP and the fragments that were used to express AP fusion proteins. b, COS-7 cells were cultured with transfection of human NgR1 or human NgR3 expression plasmid, and their respective expressions were verified by epitope-tag immunohistology. Fifty nanomolar solutions of AP-APP607, AP-APP579, and Aβ28-AP were allowed to bind to transfected cells for 2 h before washing, fixation, heat inactivation of endogenous AP, and enzymatic detection of bound fusion protein. Note the dark reaction product by arrowhead derived in cultures expressing NgR1 but not NgR3. An untransfected cell is labeled by the arrow. This is one of five experiments with similar results. c, d, Binding of AP-Aβ28, APP579, and APP607 to NgR1-expressing COS-7 cells from an experiment as in a was measured as a function of ligand concentration from 5 to 1250 nm. Data are means ± SEM from one of three experiments with similar results. The calculated Kd is noted in d. e, Purified NgR-ecto(310)-Fc, MAG-Fc, or BSA (100 ng of each protein) was coated onto microtiter wells. After blockade of nonspecific sites with excess BSA, the wells were exposed to 50 nm AP, AP-Nogo 33, or AP-APP607. Data are the means ± SEM from three similar experiments. Note the selective binding of either Nogo(1–33) or APP607 to NgR-coated wells. f, The binding of 100 nm biotin-Aβ1–40 or biotin-Aβ40–1 to immobilized NgR(344)ecto-Fc or IgG (100 ng of each protein) was detected by retention of streptavidin-AP.