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Fig. 8.

ID
ZDB-IMAGE-230420-21
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Figures for Liu et al., 2022
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Figure Caption

Fig. 8.

AKT suppresses the polyubiquitylation of zEtv2 and zScl. (A) zEtv2 and zScl degrade through the ubiquitin-proteasome pathway. Flag-tagged β-catenin, zEtv2 and zScl were co-expressed with Ub-K48R/G76A, a dominant-negative form of ubiquitin, in HEK293T cells. Lysates were then immunoblotted with the indicated antibodies. (B,C) AKT1 phosphorylates zEtv2 and zScl on specific serine residues to suppress their polyubiquitylation. HEK293T cells were transfected with the indicated constructs and incubated with 20 μM MG132 for 5 h before lysis. Ubiquitylated proteins were isolated by immunoprecipitation and ubiquitylation signals were detected by immunoblotting with an anti-HA antibody. Overexpression of wild-type (WT) AKT1, but not its kinase-deficient (KD) mutant, reduced the polyubiquitin modifications in zEtv2 and zScl proteins (B). Meanwhile, ectopic expression of AKT1 had a much more evident effect on the polyubiquitylation of the phosphorylation-resistant mutants of zEtv2 and zScl (zEtv2-S296A and zScl-S222A) compared with that of the relevant wild-type (WT) proteins (C). (D-F) Overexpression of either zEtv2/zScl-WT or their S-D form proteins in cxcr4a−/− mutants partially rescued tie1 expression in PAAs 5 and 6. Wild-type (WT) or cxcr4a−/− mutant embryos were injected with 200 pg of zEtv2/zScl-WT or their S-D or S-A form mRNA and AS-Flag-photo-MO at the one-cell stage. After treatment with 365 nm UV for 10 min at 30 hpf, the injected mRNA was overexpressed in the embryos along with AS-Flag-photo-MO degradation, and embryos were collected at 48 hpf for in situ hybridization (D). Scale bars: 50 μm. The percentages of embryos with defects in tie1 expression are shown in E,F.

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