Fig. 3. Phase separation conditions of prf.GLFG_52×12.

a Illustration of the LCST-type phase separation in this study: At a low concentration, prf.GLFG_52×12 (representing Nup98 FG domains) remains soluble in an aqueous solvent and a single phase was observed. When its concentration is increased to above a threshold termed as saturation concentration, C_sat, the amount of protein in the system exceeds the solvation capacity of the solvent and a synergistic assembly of protein molecules occurs spontaneously, leading to the formation of a protein-rich “FG phase”, which separates from the aqueous phase. FG phase is in form of scattered micrometre-sized spherical particles (“FG particles”). The solvation capacity of the solvent and thus the saturation concentration of the FG domain decreases when the temperature increases. Therefore, phase separation may also occur when the temperature is increased to above a threshold, i.e., transition temperature. After phase separation has occurred, the aqueous phase still contains solvated FG domain molecules at a concentration equal to C_sat. Right: a phase-contrast microscopy image showing a typical phase-separated state. b, c Dilutions of prf.GLFG_52×12 of indicated concentrations were prepared on ice in buffers containing 20 mM NaPi pH 6.8 and either 75 mM (b) or 300 mM NaCl (c). Each dilution was analysed by Dynamic Light Scattering (DLS) with increasing temperature until a sharp increase of light scattering intensity was observed, which indicates phase separation. The temperature at which phase separation had just occurred was taken as the transition temperature, T. d DLS analyses were repeated at indicated NaCl concentrations (C_NaCl) to determine T, which was plotted against [prf.GLFG_52×12]. Measurement for each condition was repeated three times with independent samples, and mean values are shown with S.D. as the error bars to show the variation between replicates. Each dataset for a given C_NaCl was fitted to a simple exponential function and the best fits were shown as dashed lines. e 20 μM dilutions of prf.GLFG_52×12 were prepared in buffers containing the indicated concentration of NaCl and centrifuged at the same temperature (27 °C/300 K). SDS samples of the obtained pellets (FG phase), if there were, and supernatants (soluble content) were loaded for SDS-PAGE at equal ratio (7%), followed by Coomassie blue staining for quantification. Saturation concentrations, C_sat, for individual conditions were equal to the concentrations of the supernatants. (For reader’s convenience, note that C_sat can be quickly estimated from the band intensities shown here: C_sat = ratio of supernatant/(supernatant + pellet) × assay concentration, 20 μM). C_sat was plotted against C_NaCl. Measurement for each condition was repeated four times with independent samples, and mean values are shown with S.D. as the error bars. The mean values were fitted to a simple exponential function (dashed line). f 10 μM dilutions of indicated Nup98 FG domain homologues from different eukaryotic species (Tetrahymena thermophila, Tt; Arabidopsis thaliana, At; Branchiostoma floridae, Bf and Xenopus tropicalis, Xt) were prepared in a buffer containing 75 mM of NaCl and centrifuged at the indicated temperatures. SDS samples of the obtained pellets and supernatants were loaded for SDS-PAGE at equal ratio (7%), followed by Coomassie blue staining. The exact saturation concentrations were not determined. This experiment was repeated two times independently with similar results, and the representative gel images are shown.