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. 2020 Nov 4;11:5574. doi: 10.1038/s41467-020-19211-z

Fig. 1. Bis-ANS induces LLPS of the TDP-43 LCD.

Fig. 1

a Chemical structure of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS, 1). b Representative fluorescence microscopy images for TDP-43 LCD (5 μM) in the presence of different concentrations of bis-ANS. c Representative turbidity traces for multiple protein concentrations as a function of bis-ANS to TDP-43 LCD molar ratio (n ≥ 3 technical replicates across three separate preparations for 5 μM protein, n = 3 technical replicates for 2.5 and 10 μM). d Turbidity contour plot (phase diagram) depicting the relationship between bis-ANS and TDP-43 LCD concentration. Data points used for contouring are shown in black. e Colocalization fluorescence microscopy images of liquid droplets formed by TDP-43 LCD in the presence of bis-ANS at a ratio of 2:1. Protein solution was doped with Alexa Fluor 594-labeled TDP-43 LCD (100 nM). f Representative fusion events between TDP-43 liquid droplets formed in the presence of bis-ANS. Experiments in (e, f) were performed using untagged (shown) and His-tagged protein with similar results. g FRAP of bis-ANS-containing droplets (n ≥ 14 individual droplets across three independent experiments) depicting rapid recovery within 45 s. Experiments were performed immediately after droplet formation and at 3 h. h Droplets formed by bis-ANS and TDP-43 LCD are disrupted at high temperatures (n = 3 technical replicates). All experiments were performed using a 20 mM potassium phosphate buffer, pH 6.0. Scale bars: 10 μm. All data are presented as mean values ± standard deviation.