Fig. 2

Generation of stabilized A11+ oligomers (Ab*56) for mechanistic evaluation of proteasome inhibition. a Size exclusion chromatography (Superose 6 GL 10/300) run that was used to generate a pure Aβ*56 oligomer preparation (blue solid line, left axis), with proteasome activity (amc hydrolysis) from 1 μl of the corresponding fractions (bars, right axis; as in Fig. 1), and A11 dot blot (bottom panel). b Native-PAGE Aβ*56 peak fraction from a followed by western blot using the A11 anti-oligomer antibody. c Proteasome activity (LLVY-amc hydrolysis) with titrating Aβ*56; the IC₅₀ is ~0.22 μM Aβ (4.5 kDa monomeric mass) or 18 nM Aβ*56 oligomer complexes (56 kDa mass each). d Representative raw data of proteasome activity assay (nLPnLD-amc hydrolysis) with 1.5 μM Aβ*56 oligomers (data point from c). e Change in polarization of FITC-labeled-casein protein (due to cleavage) in the presence of the 20S proteasome, with and without 10 μM Aβ*56. f Rate of FITC-casein degradation (ΔmP/min). g Representative negative stain electron microscopy image of purified Aβ*56 oligomers. Scale bar is 25 nm. h Representative tapping mode atomic force microscopy topography image of Aβ*56 oligomers. Scale bar is 0.5 μm. Heat map for oligomer height is shown on the right. i ThT fluorescence of the indicated Ab preparations. j, k Dot blot with A11 antibody i and oligomer native gel electrophoresis visualized by Coomassie stain k of non-crosslinked and glutaraldehyde crosslinked (CL) Aβ*56 oligomers before and after 4-week incubation at 4 °C. l Proteasome activity (nLPnLD-amc hydrolysis) in the presence of CL and non-crosslinked Aβ*56 oligomers (0.75 μM). Concentration of Aβ*56 oligomers is calculated based on the mass of peptide monomer (Aβ, 4.5 kDa). All controls contained an equal volume of buffer identical to that of the respective aggregates. Proteasome activity was calculated as in Fig. 1a. The data are representative of three or more independent experiments performed in triplicate. Error bars represent ± standard deviation. All following experiments utilize CL Aβ*56 oligomers unless indicated otherwise