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. 2021 Sep 23;4:1124. doi: 10.1038/s42003-021-02624-x

Fig. 5. Interactors of pre-formed fibrils of alpha-synuclein.

Fig. 5

a Schematic representation of the experimental layout for production of pre-formed α-SYN fibrils. Purified cysteine mutated α-SYN was labelled with AlexaFluor dye 594 (red spheres) and mixed with purified WT α-SYN (blue spheres). After shaking at 45 °C, fibrils were primed with the cell-free expression reaction of LTE for all the LB proteins tested. After cell-free expression, the mixture was diluted 1000-fold to reach single particle concentrations for TCCD experiments. b Schematic representation of the confocal volumes during measurements of 300 s traces. GFP monomer served as a negative control and a known interactor of αSYN as a positive control (DNAJB6) for acquisition of Association Quotients. c Bar plot of Q values for all the quadruplicated 300 s traces and normalized heatmap of binding indexes (BI = 0 for GFP monomer and BI = 1 for the highest Q value). Error bars represent mean ± SEM. Dunnett’s multiple comparisons test against traces of Fibrils + GFP. #p ≤ 0.0001, †p ≤ 0.001, ‡p ≤ 0.01, ¥p ≤ 0.05. d Traces of three of the main interactors, as defined by the top percentile in the distribution of Q values. For all traces in this figure, GFP signal is represented in positive axis, mCherry signal in negative axis.