Fig. 5

Phosphorylation modification of the Y451 site leads to the accumulation of AGPS in prostate cancer cells. a Western blot analysis of AGPS expression after MG132 (20 μM), λpp(1U/ml,2 h), or a combination. b Western blot analysis of ectopically expressed Flag-tagged AGPS protein in DU145 cells transfected with AGPS followed by treatment with λpp(1U/ml,2 h). c Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h or λpp (1U/ml,2 h). d Co-IP analysis of binding of Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells treated with λpp(1U/ml,2 h). e Western blot analysis of ectopically expressed Flag-tagged AGPS protein in DU145 cells transfected with Flag-tagged WT AGPS or mutant AGPS with or without λpp (1U/ml,2 h) treatment. f Alignment of amino acid sequences of the F443-F455 region of the AGPS protein. g Schematic diagrams depicting the potential binding and Z score of ADPS and MDM2 protein binding after AGPS Y451 site mutation to A. h Western blot analysis of ectopically expressed Flag-tagged AGPS protein in DU145 cells transfected with wild-type AGPS or AGPS Y451A mutant followed treatment with MG132 (20 μM) for 8 h. i Western blot analysis of AGPS protein expression in DU145 cells after ectopically expressed wild-type or mutant AGPS followed by treatment with CHX (50 μg/mL) for 0-, 4-, 8-, 12-, 16-, or 20- h. j Protein bands were quantified and normalized to the band intensity at the 0-h time point. * P < 0.05, ** P < 0.01. k 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. l 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