Fig 2: Attenuating phosphorylation of Ser168 by Cdk16DN or pHwa antibody disrupts the axis-inducing activity of Hwa.a Residue conservation analysis of human CDK2/16 and zebrafish Cdk2/16 proteins. The red box indicates the conserved, functionally critical aspartic acid (D). b Immunoblotting of pHwa in HEK293T cells transfected with Hwa-Falg, wild-type Cdk16, and different doses of dominant negative Cdk6 (Myc-Cdk16DN). c Quantifications of relative pHwa levels in HEK293T cells treated as in (b), N = 3.Total Hwa protein was used as an internal control. d The WISH results of boz and chd in cdk16DN mRNA injected embryos at 4 hpf and 6 hpf, respectively. e The statistical results of embryos treated as in (d). f Effect of coexpression of cdk16DN and ccnyl1-HA mRNA with hwa mRNA in Mhwatsu01sm embryos, N = 3. g Phenotypes of wild-type embryos injected with pHwa antibody or IgG at different doses, N = 4. Scale bars, 100 μm; V, ventralized; N, normal; D, dorsalized. Total Hwa proteins were used as references for quantification in (c). c A two-tailed unpaired t-test was performed and data were presented as mean ± SD. e–g A two-tailed Fisher’s exact test was performed to evaluate differences between treatments (all phenotypes were divided into two groups: Unchanged and Changed). 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.

Fig 3: Ser168 of Hwa can be phosphorylated by multiple kinases in HEK293T cells and in vitro.a Immunoblotting of pHwa from HEK293T cells cotransfected with Ccnyl1-HA and wild-type and the kinase-dead form of Myc-Cdk16(K222R). b Coimmunoprecipitation of Hwa-Flag with Myc-Cdk16, N = 3. c Immunoblotting of cytosolic/active β-catenin in HEK293T cells transfected with Hwa-Flag (WT, S168A) alone or with Myc-Cdk16/Ccnyl1-HA. d Immunoblotting of pHwa from HEK293T cells transfected with Hwa-Flag and Flag-mGSK3β (membrane-tagged GSK3β). e Coimmunoprecipitation of Myc-Cdk2 with different forms of Hwa-Flag proteins (WT, S168A and ΔPPNSP), N = 3. f Immunoblotting of pHwa from HEK293T cells transfected with wild-type and the kinase-dead form of Myc-Cdk2(T160A, K33R). g Quantifications of relative pHwa levels in HEK293T cells treated as in (a), N = 4. h Quantifications of relative cytosolic/active β-catenin levels in HEK293T cells treated as in (c), N = 4. i Quantifications of relative pHwa levels in HEK293T cells treated as in (d), N = 3. j Quantifications of relative pHwa levels in HEK293T cells treated as in (f), N = 3. k In vitro phosphorylation of purified Hwa by recombined CDK16/CCNY proteins in the absence or presence of ATP. l In vitro phosphorylation of purified Hwa by recombinant His-GSK3β in the absence or presence of ATP. m In vitro phosphorylation of purified Hwa by recombinant His-CDK2 proteins in the absence or presence of ATP. n–p Quantifications of relative pHwa levels in in vitro phosphorylation experiments treated with different kinase proteins as shown in (k–m), N = 3. The arrow and arrowhead indicate Hwa and kinase proteins, respectively (k–m). Total Hwa (a, d, f and k–m) and α-tubulin (c) proteins were used as references for quantification in immunoblotting. (g–j and n–p) A two-tailed unpaired t-test was performed and data were presented as mean ± SD. N, number of biological replicates; 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.