Extended Data Fig. 2: Phase separation property of EB1 is evolutionarily conserved in eukaryotic cells.
From: Phase separation of EB1 guides microtubule plus-end dynamics
(a) Sequence alignment of EB1 from yeast to human. The residues with positive charge were highlighted. (b) Schematic representation of the domain structures of human EB1, fission yeast Mal3 and budding yeast Bim1. (c) In vitro phase separation assay. Representative fluorescence images of Mal3-GFP phase separation in BRB80 buffer with various concentrations of KCl. Scale bar, 5 µm. (d) Representative SDS-PAGE gel of EB1 solubility in solution. Samples of EB1-GFP protein at various concentration were centrifuged as described under “Methods”. Equal volumes of supernatant (S) and pellet (P) fractions were resolved by electrophoresis and resolved proteins were visualized by CBB staining. (e) Determination of a saturation concentration (csat) of EB1-GFP. The absorbance at 340 nm (condensed protein has a distinctly different absorbance profile, leading to a pronounced uptick in A340 absorbance) was measured. The tendency of soluble and condensed concentration were labeled by red line, and the intersection was measured as csat. Data represent mean ± s.e.m. from three independent experiments. (f) In vitro phase separation assay. Representative fluorescence and DIC images of EB1-GFP (20 μM) phase separation with or without 5% 1,6-Hexanediol (Hex) in BRB80 buffer with 150 mM KCl. Scale bar, 5 µm. (g) Schematic illustration of an ultra-coarse-grained modeling for CH domain, EBH domain, the IDR region and C-terminal region. (h) Representative image of EB1 phase separation based on the ultra-coarse-grained modeling.
