Extended Data Fig. 4: Lysine/arginine residues on IDR are essential for EB1 phase separation.
From: Phase separation of EB1 guides microtubule plus-end dynamics
(a) Characterization of recombinant purified proteins used in in vitro phase separation assay. Recombinant EB1 proteins were subjected to electrophoresis followed by CBB staining. (b) Diagram of EB1 wild type and IDR chimeras. Their phase separation capacities at 10 μM were also annotated. “−” no phase separation events were observed; “+” phase separation events were observed. (c) Representative images of EB1 chimeras in COS-7 cells (Upper) and corresponding phase separation test in vitro (Lower). Scale bar, 10 μm. (d) Sequence alignment of EB1-IDR and Pub1-IDR. The residues with positive charge were highlighted. (e) Summary of in vitro phase separation behavior of EB1 RDI and KR6A mutants. EB1 mutants were purified and assessed for in vitro phase separation assay. The saturation concentration was measured by turbidity assay at indicated salt conditions. The red circles indicate phase separation (above saturation concentration), and the blue circles indicate no phase separation (below saturation concentration). (f) EB1 binding with purified +TIPs in vitro. Purified GST- or MBP-tagged +TIPs were used as affinity matrices to absorb His-EB1 wild type or KR6Q mutant as in Fig. 2k. The bound His-EB1 protein was assessed by Western blotting using an anti-His antibody.
