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. 2023 Aug 18;42(39):2878–2891. doi: 10.1038/s41388-023-02809-0

Fig. 5. PKA-dependent phosphorylation is pivotal for FBXO28-mediated SNAI2 degradation.

Fig. 5

A Huh7 cells transiently expressing FBXO28-Flag were treated with DMSO or 0.2 μM H89, 4 μM CHIR-99021, 5 μM CID755673, 0.5 μM PF-3758309, 2 nM Dinaciclib for 24 h, followed by IB analyses. B Huh7 cells transiently expressing FBXO28-Flag or a control vector were treated with DMSO or 0.5 μM H89 for 24 h prior to IB analyses. C HEK293T cells were transfected with indicated plasmids for 24 h, and then treated with DMSO or 0.2 μM H89 for 24 h, followed by IP and IB analyses. D HEK293T cells were transfected with indicated plasmids for 48 h prior to treatment with DMSO or 0.2 μM H89 for 24 h. The polyubiquitylated proteins were purified by Ni-NTA beads and detected with anti-HA antibody. E Huh7 cells expressed FBXO28-Flag or a control vector for 24 h were treated in the absence or presence of 0.5 μM H89 or 1 μM KT5720 for another 24 h, followed by transwell assays. Scale bar: 10 μm. Data were presented as mean ± SD (n = 3). **P < 0.01 and ***P < 0.001 (one-way ANOVA). F Illustration of conservative amino acids sequences in the C-terminal of SNAI2 across different species using UniProt dataset. Predicted phosphorylation sites by PKA are highlighted. G, H HEK293T cells were transfected with indicated plasmids for 48 h and subjected to IP and IB analyses in (G) or Ni-NTA beads purification in (H). The asterisks represent non-specific band. I Huh7 cells stably expressing FBXO28-mGFP or a control vector were transfected with SNAI2-HA or SNAI2 S3A-HA for 48 h and treated with CHX for the indicated time interval, followed by IB analyses. J Huh7 cells were expressed with SNAI2 S3A-HA or a control vector for 24 h prior to transfection with FBXO28-Flag for another 24 h, followed by transwell assays. Scale bar: 10 μm. Data were shown as mean ± SD (n = 3). *P < 0.05 (one-way ANOVA).