Figure S3.

Colocalization of Axin with APC, GSK3β, and β-catenin in HEK293T cells and interaction of Axin1 or its deletion mutants with APC, GSK3β, CK1α, or β-catenin. (A) Confocal fluorescence images showing the subcellular localization of dAPC2-mCherry, β-catenin-mCherry, GSK3β-mCherry, and CK1α-mCherry in Axin1 KO HEK293T cells. The nuclei were counterstained by DAPI (blue). (B) Confocal fluorescence images showing colocalization of Axin1-EGFP with dAPC2-mCherry, β-catenin–mCherry, CK1α-mCherry, or GSK3β-mCherry in Axin1 KO HEK293T cells. Graphs show the fluorescence intensity profiles along the line indicated by the arrow in the merge panels. The distance along the arrow is shown on the x axis, and the corresponding fluorescence intensities are shown on the y axis. The overlapping EGFP and mCherry signals indicate colocalization. (C) Colocalization of Axin1-EGFP with dAPC2-mCherry, β-catenin–mCherry, or GSK3β-mCherry. (B) After 12 h with or without Wnt3a conditional medium, stimulation in Axin1 KO HEK293T cells was quantified. n = 100, with three biological replicates. (D) No droplets were observed with purified β-catenin, GSK3β, or CK1α protein in 20 mM Hepes, pH 7.4, 1 M NaCl, and 10% PEG8000. (E) Differential interference contrast (DIC) images of droplets formed by 1 µM Axin1 protein and the turbidity of Axin1 LLPS were measured in the solution for the in vitro phosphorylation assay, containing 2.5 µM CK1α, 50 nM GSK3β, and 1.5 µM β-catenin and Axin1 (0.5 µM, 1 µM, and 2.5 µM) with or without 2.5% PEG8000. The droplet number was counted from three independent DIC fields, and the quantification data are shown as mean ± SD (n = 3). Statistical analysis was performed with a two-tailed unpaired t test (***, P < 0.001). (F) Confocal fluorescence images showing colocalization of Axin1-mCherry, AD1-mCherry, AD2-mCherry, and AD3-mCherry with dAPC2-EGFP, and Axin1-EGFP, AD1-EGFP, AD2-EGFP, and AD3-EGFP with GSK3β-mCherry or β-catenin–mCherry in Axin1 KO HEK293T cells. (G) FRAP analysis of dAPC2-EGFP, GSK3β-mCherry, and β-catenin–mCherry puncta when Axin1-mCherry, AD2-mCherry or Axin1-EGFP, AD2-EGFP were coexpressed. Quantification data are shown as mean ± SD (n = 3). (H) Axin1 KO HEK293T cells transfected with Axin1-EGFP were treated with or without Wnt3a conditional medium for 24 h, and then harvested for anti-GFP immunoprecipitation and anti-GSK3β, anti-CK1α, anti–β-catenin, or anti-APC immunoblotting. (I) Axin1 KO HEK293T cells (KO1, a different KO line from the one shown in Fig. 4 A) transfected with TopFlash-luciferase and Axin1-EGFP WT or mutants were treated with or without Wnt3a conditional medium for 24 h and then harvested for luciferase determination. The expression level of Axin1 WT and mutant proteins in the reporter assay was detected with immunoblotting. Data are shown as mean ± SD (n = 3). Statistical analysis was performed with a two-tailed unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (J) The expression level of the HA-tagged Axin1 WT and mutants in the fertilized Xenopus eggs (Fig. 4 C). The expressed proteins are indicated with an asterisk. Scale bars in A, B, and F, 10 µm; in D, E, and G, 2 µm. IB, immunoblot; IP, immunoprecipitation; Vec, vector; TCL, total cell lysate.