Figure 3. A–B: Biochemical analysis of the Dounce-derived soluble fractions.

The presence of APP derived fragments, such as Abeta (6E10) and soluble APPalpha (extracellular), together with LC3-I (cytosolic), ATPsynthase-beta (mitochondrial) and LC3-II (autophagic vesicles) was tested by western blots. A representative western blot (A) or quantitative analysis of three experiments (B) is shown. Although contaminated with cytosolic proteins (LC3-I), the TBS soluble fraction was enriched in extracellular soluble APP-alpha and lacked monomeric Abeta. The monomeric Abeta was predominantly concentrated in vesiculated, LC3-II positive, and plaque associated fractions. C: Dounce homogenization preserved the integrity of Abeta plaques. After a first homogenization and centrifugation, pellets containing Abeta plaques were re-homogenized using Dounce or sonication (4 pulses) and the soluble Abeta content assayed using western blots and 6E10. Monomeric Abeta was exclusively observed after sonication.