Fig. 3: Rph3A undergoes phase separation in a cell-free system.

a The images of EGFP alone (left), the full-length Rph3A fusion protein at a concentration of 10 μM in buffer containing 2% PEG8000 (middle) or a concentration of 50 μM without the crowding reagent (right). b Representative images and size data for droplets composed of the Rph3A fusion protein at different concentrations. The data are displayed as the mean ± SEM (20 μM: n = 145 droplets; 10 μM: n = 125 droplets; 5 μM: n = 72 droplets; 2 μM: n = 28 droplets, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). c Representative images and size data for droplets composed of the Rph3A fusion protein at 10 μM in buffer with different salt concentrations. The data are displayed as the mean ± SEM (150 mM: n = 72 droplets; 200 mM: n = 31 droplets; 250 mM: n = 6 droplets, 300 mM: no droplets, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). d Fusion events of droplets formed by the Rph3A fusion protein. Time-lapse images of fusing droplets are showed. e Representative images and quantification of fluorescence recovery from the FRAP analysis of droplets composed of the Rph3A fusion protein. The data are displayed as the mean ± SEM (n = 7 droplets). Scale bar, 1 μm. f Schematic illustration of recombinant Rph3A-EGFP fusion proteins. g Representative images and size data for droplets composed of full-length and truncated Rph3A fusion proteins at different protein concentrations. The data are displayed as the mean ± SEM (20 μM: n = 122 droplets (FL); n = 101 droplets (IDR1); n = 17 droplets (ID1R9A), 10 μM: n = 114 droplets (FL); n = 120 droplets (IDR1); n = 5 droplets (ID1R9A), 5 μM: n = 54 droplets (FL); n = 60 droplets (IDR1); no droplets (ID1R9A), no droplets in EGFP group, ^*p < 0.05, ^**p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test). Source data and p values of b, c, e and g are provided in the Source Data file.