FIGURE 2.

Formation of sarkosyl-insoluble fibrillar aggregates of TDP-43^FL proteins. A, aggregation kinetics of 1 μm refolded TDP-43^FL proteins monitored by the increase of solution turbidity at 405 nm: open circles, WT; filled circles, M337V; open squares, A382T. Three independent experiments were performed to estimate errors. Error bars indicate S.E. B, formation of TDP-43^FL aggregates with limited solubility in a buffer containing 1% sarkosyl. After the turbidity changes reached plateau (∼1,600 min in A), ultracentrifugation separated the soluble supernatant (lane 1, soluble fraction) from insoluble pellets, which were resuspended and sonicated in PBS containing 1% sarkosyl with the same volume as that of the corresponding supernatant. Ultracentrifugation again separated supernatant (lane 2, sarkosyl-soluble fraction) from the insoluble pellets, which were resolubilized in PBS containing 2% SDS with the same volume as that of the corresponding supernatant (lane 3, sarkosyl-insoluble fraction). Equal volumes of the samples were boiled in the presence of β-mercaptoethanol, loaded on a 12.5% SDS-PAGE gel, and stained with Coomassie Brilliant Blue. C, 1 μm TDP-43 before (open bars) and after (∼1,600 min in A, filled bars) aggregation was mixed with 25 μm thioflavin T. Fluorescence intensity was measured as described under “Experimental Procedures.” Three independent experiments were done to estimate errors. Error bars indicate S.E. D–F, electron micrograms of TDP-43^FL aggregates: WT (D), M337V (E), and A382T (F). The bar in each panel represents 50 nm.