Fig. 2. Mutation at Ser168 of Hwa attenuates the activation of β-catenin signaling.
a The left panel indicates the classification of rescued phenotypes at 24 hpf (V2 < V1 < N < D1 < D2; V, ventralized; N, normal; D, dorsalized); the right panel shows the rescue efficiency of wild-type and single-amino acid-substituted hwa-HA mRNAs in Mhwatsu01sm/tsu01sm embryos, with 5 pg of each hwa-HA mRNA injected per embryo at the 1-cell stage, N = 2. b Immunoblotting of β-catenin from the cytosol (active form) and total cell lysate (TCL) of HEK293T cells overexpressing wild-type or mutant Hwa-HA protein. PPNSP motif deletion or Ser168 mutation nearly abolished the activation of the β-catenin signal by Hwa. c Quantifications of relative cytosolic β-catenin levels in HEK293T cells treated as in (b), N = 3. d SuperTop Flash was applied to check the β-catenin signal-inducing activity of wild-type and Ser168 mutant Hwa (S168A and S168E), N = 3. e Coimmunoprecipitation of HA-TNKS1 with wild-type and mutant Hwa-Flag proteins. Protein lysates were immunoprecipitated with anti-Flag antibodies; the arrow indicates the HA-TNKS1 protein, N = 3. f Immunoblotting of HA-Axin1 in cells co-transfected with different doses of wild-type or S168A Hwa-HA plasmids. The arrow indicates the HA-mAxin1 protein. g Quantifications of relative HA-Axin1 protein levels in HEK293T cells treated as in (b), N = 3. α-tubulin (b) or total HA-TNKS1 (f) was used as references, and relative protein levels are indicated in (c) and (g). a A two-tailed Fisher’s exact test was performed to evaluate differences between treatments (all phenotypes were divided into two groups: Unchanged [V2] and Changed [V1-D2]). (c–g) A two-tailed unpaired t-test was performed and data were presented as mean ± SD. N, number of biological replicates; n, total number of embryos in each treatment; Significant differences are indicated by ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Source data are provided as a Source Data file.