Figure 8

Detection of human Aβ peptides in neurosphere culture supernatants. (A) Sixty microgram of total protein from 80 to 100-fold concentrated neurosphere culture supernatant were size fractionated in Tris-Glycine-SDS-PAGE along with monomeric and oligomeric Aβ42 peptides as positive controls, transferred onto 0.2 μm PVDF membrane and immunoblotted with 6E10 antibody after heat induced epitope retrieval (HIER). Chemiluminescence digital images show the presence of Aβ monomers and various oligomeric Aβ peptides only in Tg+ve but not in Tg-ve neurosphere culture supernatants. Molecular weight analysis of these bands was compatible with the oligomers indicated. (B) A longer electrophoresis of the same samples resolved high molecular weight oligomers as potential Aβ 10-mers and 12-mers. (C) Densitometric analysis was used to measure the monomeric Aβ peptides in NS culture supernatants employing known amounts of Aβ42 as standards. (D) Densitometric analysis of Aβ oligomers (2-mer to 64 kDa) is presented as histograms of mean + standard deviation. Data represents four independent experiments. LO = Large Oligomer.