(A) In vitro binding of FcγRIIb-ED protein to cell-derived Aβ. Purified GST or hFcγRIIb-ED protein was incubated with CHO- or 7PA2-CM for 6 hours and then immunoprecipitated with anti-GST or anti-FcγRIIb antibody. The precipitates were analyzed by Western blotting. L.C., light chain. (B) Direct interaction of FcγRIIb-ED with Aβ_1-42 in SPR analysis. BSA or oligomeric Aβ_1-42 was immobilized and the interactions with hFcγRIIb-ED were analyzed using Biacore 3000. (C) Interaction of FcγRIIb with oligomeric Aβ_1-42 in AD patients. Hippocampal extracts from AD patients and controls (C) were subjected to immunoprecipitation assays using IgG or Nu-1 antibody, and the precipitates were analyzed with Western blotting. Asterisk indicates nonspecific signal, which differs in size from the Aβ monomer. (D) Computational simulation showing the structure of the FcγRIIb-Aβ_1-42 complex. The N terminus of Aβ_1-42 and IgG-binding region of hFcγRIIb were handled using the Affinity program. (E) Inhibition of Aβ_1-42-induced reporter activation of the FcγRIIb-ED/CD40-Luc chimera by Aβ_1-9 or FcγRIIb_107-114. Synthetic peptides spanning the predicted interaction regions of FcγRIIb and Aβ_1-42 and their mutants are represented (top). The reporter assay was performed with or without synthetic peptides with a molar ratio of 1:3 (bottom). (F) Binding of Aβ_1-42 to the Ig2 domain of FcγRIIb. Domain structures of FcγRIIb and its deletion constructs are presented (top). HEK293T cells were transfected with the constructs and treated with 1 μM Aβ_1-42 for 1 hour. Cell lysates were subjected to immunoprecipitation analysis using Aβ antibody or preimmune (Pre), and the precipitates were analyzed with Western blotting. Expression of each FcγRIIb protein was checked in whole cell lysates. IP, immunoprecipitation.