Fig. 1.

Hexameric Aβ₄₂ derived from CHO cells expressing human APP metabolites. a Full-length APP is cleaved by β-secretase at the β site, located at the N-terminus of Aβ, to produce a 99-amino acid membrane-bound fragment (C99) encompassing Aβ and AICD. The C99 construct expressed here in the cells was fused to the signal peptide (SP) of APP. C99 is cleaved by γ-secretase to release Aβ. The C42 construct is composed of the SP of APP and the Aβ₄₂ sequence. The epitopes of the primary antibodies used in this study are indicated on the scheme; either directed against human Aβ (clones W0-2 and 6E10 targeting its N-terminal part, and Aβ₄₀ and Aβ₄₂ antibodies specifically targeting the free C-terminal end of Aβ) or against the C-terminal region of APP (APP-C-ter). Nt N-terminus, Ct C-terminus, sAPP soluble APP, AICD APP intracellular domain, EC extracellular, IM intramembrane, IC intracellular. b Detection of ~ 28 kDa assemblies by Western blotting in CHO cell lysates and culture media following expression of either C42 or C99. These assemblies are recognized by Aβ specific antibodies (such as W0-2 here), but not by the anti-APP-C-ter, suggesting they emerge by assembly of Aβ. Media samples were lyophilized and all samples were immunoprecipitated with the anti-Aβ (W0-2) antibody prior to analysis. In the media of C99-expressing cells, intermediate assemblies are also observed; monomers, dimers, and trimers. EP empty plasmid. c Isolation of cell-derived Aβ assemblies. The media of CHO cells expressing either EP, C42, or C99 were immunoprecipitated and separated using the GELFrEE™ technique. We optimized a method to collect the ~ 28 kDa Aβ assemblies as an isolated liquid fraction. Dashed lines indicate that proteins were run on the same gel, but lanes are not contiguous