Figure S2.

Related to Figure 2

A: Coomassie stained, non-native 4%–12% NuPage Bis-Tris gel showing ~1 μg of heterologous expressed and purified proteins. B: Phase diagram of G3BP1(WT) with control parameters KCl and protein concentration. Phase separation was scored by the absence or presence of G3BP1 condensates. C: Fluorescence images of G3BP1(WT)-RNA condensates before (top) and after (bottom) increasing the NaCl concentration from 30 mM to 280 mM. Scale bar, 10 μm. D: Phase diagram of G3BP1(ΔRG) as a function of protein and RNA concentration. (x) represent tested conditions and depicts that no condensates were present. E: Phase separated fraction of G3BP1(WT), G3BP1(ΔRG) and G3BP1(ΔNTF) as a function of PEG-20K concentration, in the absence of RNA (mean, SD, fit, n = 10 FOV). F: Fluorescence images of mCherry-Caprin-1 in the absence and presence of GFP-G3BP1. Scale bar, 10 μm. Right panel shows the quantification of the mCherry-Caprin-1 partition coefficient into G3BP1-RNA condensates as a function of G3BP1(WT) concentration. Caprin-1 concentration was 2.5 μM. G: EMSA to determine the apparent binding affinity of G3BP1 in the absence (gray) or presence of 1 μM mCherry-Caprin-1 (magenta). Quantification of one representative experiment is shown. H: Fluorescence images of indicated GFP-labeled client proteins partitioning into reconstituted SNAP(Alexa546)-labeled G3BP1-RNA condensates. Client proteins were added to preformed G3BP1-RNA condensates. As control, client proteins were tested in the absence of G3BP1-RNA condensates, showing that none of them phase separates (top panel) (scale bar, 10 μm). I: Fluorescent images of preformed FUS-GFP condensates (10 μM) in the presence of SNAP(Alexa546)-labeled G3BP1 (6 μM), in the absence of RNA. J: Fluorescence images of G3BP1(WT)-RNA condensates in presence of Cy3-labeled Ubc9(ts) and Ubc9(WT) (scale bar, 5 μm). Quantification of the mean average Cy3-Ubc9 fluorescence in G3BP1-RNA condensates is shown. K: Quantification of the diffusion times of Cy3-labeled Ubc9(ts) and Ubc9(WT) in solution, determined by FCS. Except for panels E and I, G3BP1-RNA condensates were formed in the presence of 75 ng/μl of total RNA (isolated from HeLa cells) and 1% PEG-20K.