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pro2339-sup-0001-suppfig1.tif886KFigure S1. Apparent disaggregase activity of PNS depends on the type of 96-well plate used for the assay and corresponds to the amount of Aβ1-40 adsorbed to the wells. (A) Incubation with 60 µg/ml mouse brain PNS leads to loss of ThT fluorescence of Aβ1-40 fibrils (15 µM) in standard plates. More Aβ1-40 is lost from Solution and recovered by 8 M GndCl Wash for samples incubated with PNS than for samples containing fibrils and PBS alone, as quantified by RP-HPLC. (B) No loss of ThT fluorescence is observed in low-binding plates, and little Aβ1-40 is lost from Solution or recovered in the 8 M GndHCl Wash for either PBS- or PNS-treated samples. The slight loss in overall amount of Aβ1-40 incubated with PNS is attributed to proteolysis. (C) Time course of adsorption of Aβ1-40 fibrils. Aβ1-40 fibrils were incubated in the absence or in the presence of 50 µg/ml PDS (upper left) or 60 µg/ml mouse brain PNS (lower left). At four time points throughout the incubation (indicated by vertical arrows), samples were processed as described in the experimental scheme of Figure 2. The amount of Aβ1-40 in Solution and Wash was determined by RP-HPLC for PDS (upper right) and PNS (lower right) experiments. Note that even after only 4-5 h incubation in the presence of PDS or PNS, we observed an increase in adsorption of Aβ1-40 to plate wells compared to PBS controls. (Error bars indicate standard deviations of three independent experiments.)
pro2339-sup-0002-suppfig2.tif113KFigure S2. Mouse brain PNS facilitates the adsorption of intact amyloid fibrils to plate wells. Aβ1-40 fibrils (pre-aggregated from 15 µM monomerized Aβ1-40) were incubated in the absence (PBS) or in the presence of 15 or 45 µg/ml mouse brain PNS. After 40 h of incubation, the plate was sonicated for 1 h, and the ThT fluorescence measured again. ThT fluorescence of PNS samples is recovered, indicating that Aβ fibrils are adsorbed to the wells.
pro2339-sup-0003-suppfig3.tif249KFigure S3. Denaturation and adsorption of the C. elegans PDS proteins. (A) Incubation of 50 µg/mL PDS alone, without Aβ1-40 fibrils, with Sypro Orange in standard plates for 22 h with agitation for 5 s every 10 min resulted in increased fluorescence over time, indicating denaturation of PDS proteins. (B) PDS alone, without Aβ1-40 fibrils, was incubated with rotary shaking (24 rpm) in Eppendorf tubes for 35 h at 37°C. The supernatants in the Eppendorf tubes were then discarded and the protein adsorbing to the walls visualized with Coomassie blue. (C) PDS alone, without Aβ1-40 fibrils, was incubated with agitation for 5 s every 10 min in 96-well plates for 35 h. The amount of protein in the Solution (S) and in Wash (W) was quantified by BCA. A significant amount of the PDS adsorbs to the well walls in the standard plates as compared to the low-binding plates.

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