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. 2016 Dec 2;27(2):202–225. doi: 10.1038/cr.2016.141

Figure 4.

Figure 4

Net1-regulated β-catenin S675 phosphorylation is required for zebrafish dorsal axis formation. (A and B) Overexpression of wild-type Net1 (A) but not Net1 loss-of-function mutants (B) results in an increase in β-catenin S675 phosphorylation. HEK293T cells transfected with plasmids encoding Flag-Net1, Flag-Net1-L266E or Flag-Net1-W437L were treated with or without Wnt3a CM. Lysates were analyzed by western blot using the indicated antibodies. (C) Knockdown of net1 leads to a significant decrease in β-catenin S675 phosphorylation. HEK293T cells were transfected with net1 shRNA expressing plasmids and treated with or without Wnt3a CM before immunoblotting. (D) HEK293T cells were co-transfected net1 shRNA and Net1 plasmids, then treated with Wnt3a CM. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Note that only wild-type Net1 can rescue the relative decrease in β-catenin S675 phosphorylation in net1-depleted cells. (E and F) Western blots of total lysates from wild-type embryos, 4 ng cMO or net1 MO1-injected embryos, and net1 morphants co-injected with 200 pg mRNAs encoding wild-type net1 or net1 mutants. The phosphorylation level of β-catenin S675 is upregulated around MBT (1 K-cell stage) in wild-type embryos (E, left panels), while this relative increase in β-catenin S675 phosphorylation is abolished in net1 morphants (E, right panels). β-cat, β-catenin. Only overexpression of wild-type Net1 can rescue the decrease in β-catenin S675 phosphorylation in net1 morphants (F). In panel A-F, quantification is the relative density of phospho-specific signals to corresponding total protein signals (mean ± SD, three independent biological repeats). (G) The phosphorylation of β-catenin S675 is essential for dorsal gene expression. The expression of boz and chd was examined at the sphere stage in embryos injected with the indicated MOs and mRNAs. Injection dose: β-catenin S675D mRNA, 150 pg; β-catenin S675A mRNA, 400 pg; net1 MO1, 4 ng. (H) Phosphorylated β-catenin has stronger ability to enhance dorsal gene expression. The expression of boz and chd was examined at the sphere stage in embryos injected with the 150 pg indicated mRNAs. (I) Phosphorylated β-catenin induces much earlier expression of boz and chd. The expression of boz and chd was examined at the 512-cells stage and High stage, respectively, in embryos injected with 150 pg indicated mRNAs.