Figure 5.

Aβ dimers in lipid rafts from Tg2576 and human brains. A, Fractions 1–11 from 11-month-old Tg2576 brain. Ba, Fraction 4 from 4-, 8-, 10-, and 12-month-old Tg2576 brains. Bb, Fraction 4 from 5-, 6-, 7-, 8-, 10-, and 11-month-old Tg2576 brains labeled with 4G8. The Aβ in A and B was immunoprecipitated with polyclonal antibody 3160 and then immunoblotted with 4G8, a monoclonal antibody that detects both monomers and dimers. Dimers were the predominant form of Aβ detected in lipid rafts. Aβ dimers were detected at 6 months and progressively accumulated thereafter. C, Epitope mapping of fraction 4 from 17-month-old Tg2576 brain using anti-Aβ and anti-APP antibodies. Aβ was immunoprecipitated with 3160 and then immunoblotted with Z31preA (B), BAN-50 (C), 4G8 (D), BA-27 (E), BC-05 (F), and O443 (G). Lane A is synthetic Aβ40 (100 pmol) immunoprecipitated with 3160 and detected by BAN-50. The 8 kDa Aβ dimer and the 4 kDa Aβ monomer were detected by all anti-Aβ antibodies. BA-27 specifically detects the C terminus of Aβ40 and BC-05 is selective for the C terminus of Aβ42, so the labeling by these antibodies shows that the 8 kDa protein terminates at Aβ40 or at Aβ42 on the carboxylside. Z31preA, which recognizes the APP epitope just amino to Aβ failed to detect the 8 kDa band, showing that the 8 kDa band is dimeric Aβ and not an Aβ-bearing APP fragment that extends past Aβ on the amino side. The 8 kDa Aβ dimers were also not detected by anti-C terminus of APP (O443). Because 3160 recognizes the N-terminal region of Aβ, p3 was not detected with this immunoprecipitation. D, Fractions 1–11 from AD, pathological aging (PA), and control brains. The Aβ in the fractions shown in D was directly immunoblotted with 4G8.