FIG. 6. Reversible interactions between Hsp70 and α–Syn.

A, immunoprecipitation of α–Syn from inhibition reactions. Five μg of α–Syn was immunoprecipitated from Hsp70-containing reactions at 0, 24, and 72h after incubation using Syn211 antibody. Total input (T), flow through (F), and immunoprecipitate (IP) representing 20%, 10% and 40% of each fraction were separated by SDS-PAGE and immunoblotted for Hsp70 (3a3) or α–Syn (Syn303). B, schematic of α–Syn⁵⁹⁴ displacement assay. Assembly reactions containing labeled α–Syn (α–Syn⁵⁹⁴) were inhibited using substoichiometric quantities (1:10) of chaperone or GST as a control. After 72 h incubation, an equal amount of fresh monomeric α–Syn⁴⁸⁸ was added to each reaction. Aggregation was assessed by measuring the relative fluorescence of α–Syn⁵⁹⁴ and α–Syn⁴⁸⁸ in the soluble fractions following centrifugation at 100,000 × g. Reactions containing GST (C) or GST-Hsp70^386-640 (D) were monitored immediately, 24, or 48 h after α–Syn⁴⁸⁸ was added. Black and grey lines denote soluble α–Syn⁵⁹⁴ and α–Syn⁴⁸⁸, respectively. Similar proportions of both α–Syn⁵⁹⁴ and α–Syn⁴⁸⁸ are found in the soluble fraction following extended incubation, indicating that previously bound α–Syn⁵⁹⁴ is readily displaceable. Data represent means from 4 independent reactions ± s.e.m.