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. 2019 Mar 21;13:216. doi: 10.3389/fnins.2019.00216

FIGURE 2.

FIGURE 2

Non-aggregated αS enhances vesicle docking activity even at excessive concentrations. (A) SDS-PAGE and western blot analysis of purified recombinant αS. A 12% SDS-PAGE gel containing αS through the two rounds of gel filtration: Lane 1 contains αS after the 1st round of gel filtration. Lane 2 contains αS after the 2nd round of gel filtration (left panel). A western blot of αS through the two rounds of gel filtration (right panel). The lane order is the same as SDS-PAGE. Bracket encloses higher molecular weight αS bands observed only in the sample from a single round of gel filtration. (B) Analysis of vesicle docking with twice purified αS (lane 2 in A). The number of docking events for each αS concentration is normalized relative to that without αS. The dashed line represents the vesicle docking activity of SNAREs without αS. Studies without VAMP2 were done by flowing v-liposomes without VAMP2 reconstituted in them along with 10 μM αS over the supported t-bilayer. No events were observed. In total 3,148 docking events from 82 independent measurements are analyzed. (C) Analysis of vesicle docking with one-time purified αS (lane 1 in A). The number of docking events from each αS concentration is normalized with respect to those without αS. The dashed line represents the docking activity of SNAREs without αS. Studies without VAMP2 were done in the same way as in B. A total of 6,249 vesicle docking events from 65 independent measurements are analyzed.