Figure 1

Oligomeric and fibrillar Aβ stimulated a qualitatively similar and dose-dependent increase in tyrosine phosphorylated protein levels. Aβ1–42 oligomers (Aβo) and Aβ1–42 fibrils (Aβf) were prepared to a 100 μM concentration via 4 degree incubation overnight (Aβo) or 37 degree incubation 7 days (Aβf) then diluted to 20 μM in DMEM/F12 media and incubated an additional 48 hours at 37 degree C to determine peptide states under bioassay conditions. 1 μg (A, C) or 0.2–2 μg (B) of peptides were separated by (A, B) 15% SDS-PAGE or (C) 15% non-denaturing gel electrophoresis and Western blotted with anti-Aβ antibody, 6E10. (D) Alternatively, prepared Aβo and Aβf were dot blotted (5 μg) each onto PVDF and blotted with anti-Aβ antibody, 6E10, or anti-oligomer antibody, A11. (E) Primary mouse microglia were unstimulated (control) or stimulated with increasing concentrations of Aβo, Aβf, or vehicle. Cells were lysed after 5 min with RIPA buffer. Cell lysates were separated by 7% SDS-PAGE and Western blotted with anti-phosphotyrosine antibody (4G10) and anti-ERK2 antibody (loading control). Antibody binding was visualized by chemiluminescence. Blots are representative of at least three independent experiments.