Fig. 2
Effects of macromolecular crowding on homotypic NPM1 and heterotypic NPM1-S6N LLPS. a Phase diagrams for LLPS of mixtures of NPM1 and S6N in the presence of different concentrations of PEG (0%, 5%, and 15% PEG, as indicated) determined by turbidity assays. Phase separation was not observed for protein concentrations represented by open circles (OD340 <0.1) and was observed for those represented by solid colored circles (OD340 ≥0.1). The solutions also contained 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer. b Confocal microscopy images of NPM1-A488 (green) and S6N-A647 (red) droplets in the presence of different PEG concentrations (0%, top; 5%, middle; and 15%, bottom); scale bar = 10 μm. NPM1 and S6N concentrations were 10 μM each represented in the phase diagrams in a by thin blue, green, and orange circles around the solid circles of similar color. c Ratios of concentrations of NPM1 and S6N within individual NPM1-S6N droplets. NPM1-SURF6 molar concentration ratios ([NPM1]/[S6N]) for droplets prepared in the presence of different concentrations of PEG (0%, top panel; 5%, middle panel; or 15%, bottom panel; n ≥ 45). The black line indicates the average [NPM1]/[S6N] ratio for droplets at the specified crowding agent concentrations. In these experiments, concentrated PEG was added to pre-mixed solutions (in 10 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5 buffer) of NPM1 and S6N to give the noted final concentrations. d FRAP curves for NPM1 for NPM1-S6N droplets prepared in the presence of different PEG concentrations (0%, blue; 5%, green; or 15%, orange) after incubation for 4 h; ROI = 1 µm circular area in the center of the droplet. Values represent mean ± s.d
