Fig. 5. Phospho-p53 (S315) facilitates FBXL6-p53 interaction.

a HEK293 cells were transfected with Flag-FBXL6 and HA-p53 plasmids for 48 h followed by MG132 treatment for 5 h. Cells were lysed in RIPA buffer (without SDS) and treated with Fast AP where indicated. p53 was immuno-precipitated with anti-HA magnetic beads and subsequently examined in IB with the indicated antibodies. b HEK293 cells were transfected with vectors expressing HA-p53 or its mutants and increasing amounts of Flag-FBXL6. Cells were collected 48 h later and examined with the indicated antibodies. β-actin was used as the loading control. c Amino acid sequences of p53 from the indicated species are highly conserved around the S315 phosphorylation site. d In vivo polyubiquitination of p53 and its mutants. HCT116 (p53^−/−) cells were transfected with plasmids expressing Myc-tagged ubiquitin, Flag-FBXL6, and HA-p53 or its mutant as indicated. Cells were treated with MG132 for 5 h before collected. Immunoprecipitation and IB analysis were performed as described in Fig. 4c. e HEK293 cells were transfected with the indicated plasmids and then treated 48 h later with MG132 for another 5 h. Immunoprecipitation and IB analysis were performed as described in (a). f GST pull-down assays were performed with the indicated GST-FBXL6 and His-p53 proteins. g Endogenous interaction of FBXL6 and phospho-p53 (S315). HCT116 and RKO cells were transfected with an expression vector encoding Flag-tagged AURKA and then after 48 h were treated with MG132 for 5 h before lysis. The lysates were then subjected to immunoprecipitation using the phospho-p53 (S315) antibody. Immuno-complexes and lysates (input) were analyzed by IB using the indicated antibodies.