Extended Data Fig. 8: Characterization of EB1 phase separation in the presence of microtubule and +TIPs in vitro.
From: Phase separation of EB1 guides microtubule plus-end dynamics
(a) Characterization of EB1 phase separation in microtubule plus-end tracking at different KCl concentrations using in vitro TIRFM assay. The concentration of EB1-GFP was 50 nM in the presence of various concentrations of KCl. Scale bar, 5 μm. (b-c) Quantitative analyses of fluorescence intensity of EB1 at microtubule plus-ends (b) and microtubule growth rate (c) in (a). For each group, n = 30 microtubules pooled from three independent experiments. Data represent mean ± s.e.m. Ordinary one-way ANOVA followed by Tukey’s post hoc test was used to determine statistical significance. ****p < 0.0001; ns (not significant) indicates p > 0.05. (d) Electron micrograph of EB1-hnRNPA1 chimeras (2 μM) from in vitro phase separation assay as carried out in Fig. 4a. Scale bar, 500 nm. (e) Representative images of co-phase separation of FITC-labeled His-EB1 (EB1-FITC) and other +TIPs co-condensates in the presence of 150 mM KCl. Scale bar, 5 μm. (f) Partition coefficients (PCs) for EB1 and other +TIPs from experiment in (e). Data represent mean ± s.e.m. from three independent experiments. Dotted line, PC = 1. (g) Quantitative analyses of droplet areas in (e). For EB1-FITC, n = 30; EB1-FITC + TIP150, n = 50; EB1-FITC + MCAK, n = 30; EB1-FITC + p150Glued, n = 30; EB1-FITC + CLIP170, n = 30; for +TIPs only, no droplet was observed in ten fields of view under microscope; n represents the number of EB1 droplets pooled from three independent experiments. Data represent mean ± s.e.m. Ordinary one-way ANOVA followed by Tukey’s post hoc test was used to determine statistical significance. ****p < 0.0001; ns (not significant) indicates p > 0.05.
