Fig. 5: AKT phosphorylates UFL1 at the T426 site to enhance its UFMylation and promote TNBC progression.

a UFMylation assay of the Flag-S-AKT1 was examined in cells treated with indicated concentrations of capivasertib. b Cells were transfected with vector or Flag-S-UFL1, followed by S-agarose pull-down. Western blotting was performed using the indicated antibodies. c Cells were transfected as indicated and treated with capivasertib (10 μM) for 24 h before harvest. Then, cell lysates were pulled down using S-agarose and western blotting was performed. d AA sequences around the T426 residue in UFL1 are conserved across different species. Arrows, threonine residues that are conserved across species. e Cells were transfected with vector, Flag-UFL1 WT or T426A mutant and cell lysates were subjected to immunoprecipitation with an anti-Flag antibody. Western blotting was performed using the indicated antibodies. f HCC1806 cells were treated with vehicle or indicated concentrations of capivasertib for 24 h, and western blotting was performed. g HCC1806 cells stably expressing control or AKT1/2/3 shRNAs were generated, and western blotting was performed using the indicated antibodies. h UFMylation assay of the Flag-S-AKT1 was examined in cells co-transfected with the indicated UFMylation system components. i Cells transfected as indicated were treated with MG132 and subjected to anti-Flag immunoprecipitation to assess UFL1 interaction with UFBP1, CDK5RAP3, or UFC1. j Cells were transfected with vector or Flag-UFL1 and treated with capivasertib (10 μM) for 24 h before harvest. Cell lysates were subjected to immunoprecipitation with an anti-Flag antibody. Western blotting was performed using the indicated antibodies. k HCC1806 cells stably expressing vector, Flag-UFL1 WT, or T426A mutant were generated, and western blotting was performed with the indicated antibodies. Cell proliferation was measured. l HCC1806 cells, as in (k), were treated with the indicated concentrations of cisplatin or doxorubicin and cell survival was determined. Western blotting is representative of three independent experiments (f, g, k). Data were presented as mean ± SD of three independent experiments (k, l). Data were analyzed by two-sided one-way ANOVA in (k). Source data are provided as a Source Data file.