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. 2021 Mar 2;220(4):e202012112. doi: 10.1083/jcb.202012112

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© 2021 Nong et al.

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Figure 1. — Axin1 undergoes LLPS. (A) Confocal images showing Axin1-EGFP puncta in Axin1 KO HEK293T cells. The nuclei were counterstained by DAPI (blue). (B) FRAP showing the recovery of the fluorescent signal in Axin1-EGFP puncta in Axin1 KO cells. (C) Droplet fusion of Axin1-EGFP puncta in an Axin1 KO cells. (D) Droplet formation of 2 µM Axin1 protein with 10% PEG8000. (E) Axin1 LLPS with 2.5% PEG8000. Left: Differential interference contrast (DIC) imaging. Right: turbidity measurement. (F) FRAP analysis of green-labeled Axin1 droplets. (G) Axin1 droplet fusion. (H) Quantification of colocalized LLPS droplets of green- and red-labeled Axin1 protein. ***, P < 0.001. n = 300. (I) Schematic diagram of Axin1 and its mutants. (J) Confocal images of EGFP-tagged proteins in Axin1 KO cells. (K) FRAP analysis of EGFP-tagged protein puncta in Axin1 KO cells. (L) Droplet formation of 2 µM Axin1 and its mutant proteins with 10% PEG8000. The droplet number was counted from three independent DIC fields. The size of droplets was measured from 200 droplets for each group. **, P < 0.01; ***, P < 0.001. (M) FRAP analysis of red-labeled Axin1 and its mutant protein droplets. Data in H, L, and M are shown as mean ± SD (n = 3). Scale bars in A and J, 10 µm; in B–D, F, G, and K–M, 2 µm. β-cat, β-catenin; del, deletion.