Figure 3.

Hsp70 impairs ASyn oligomerization in multiple assays. Various concentrations of Hsp70 were added to the (A) split gLuc (42), (B) split nLuc, and (C) FRET (42)-based ASyn oligomerization assays. All assays were as in methods. B, in total, 5 μM each of ASyn-SmBiT and ASyn-LgBiT were incubated with 1 mM ADP, 1 mM Mg. C, ASyn-Cy3 and ASyn-Cy5 were incubated with 0 (blue), 10 (red), or 30 (green) μM Hsp70 with 1 mM MgCl₂, 1 mM ADP. Triplicates (circles) are shown with first-order kinetic fit (line). Hsp70 impaired ASyn oligomerization in all three assays. The IC₅₀ of Hsp70 for each assay is calculated based on the plateau signal at 24 h using the GraphPad Prism nonlinear regression model for dose response (three parameters). D, in total, 10 μM of each of ASyn-SmBiT and ASyn-LgBiT was incubated for 24 h after which 20 μM Hsp70, 1 mM Mg, and 1 mM ADP were added, and the reactions further incubated. Hsp70 fails to disassemble already formed ASyn oligomers. Means ± s.d. shown, n = 3.