Fig. 4

AGPS binds to MDM2 at the C-terminal amino acid sequence of the F443-F455 segment. Schematic diagrams depicting a set of recombinant protein constructs for a GST-MDM2 and b GST- AGPS. c Western blot analysis of AGPS proteins in DU145 whole-cell lysate pulled down by GST or GST-MDM2 recombinant proteins. d Western blot analysis of MDM2 proteins in DU145 whole-cell lysate pulled down by GST or GST-AGPS recombinant proteins. e Schematic diagrams depicting the potential binding motif of AGPS with MDM2 protein, molecular dock with AGPS (AlphaFold: AF-O00116-F1) and MDM2(6KZU). f Western blot analysis of ectopically expressed Flag-tagged AGPS protein in DU145 cells transfected with wild-type or mutant AGPS. g Western blot analysis of AGPS protein expression in DU145 cells after ectopically expressed wild type or mutant AGPS followed by treatment of CHX (50 μg/mL) for 0-, 4-, 8-, 12-, 16-, or 20- h. h Protein bands were quantified and normalized to the band intensity at the 0-h time point. * P < 0.05, ** P < 0.01, *** P < 0.001. i Co-IP analysis of binding of Flag-tagged WT AGPS or mutant AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h. j Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged WT AGPS or mutant AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h