Figure 6.

TRIM45 mediates the K48-K63 ubiquitination transition of p53. (a) Lysates of U87 cells transfected with plasmids expressing Flag-p53, hemagglutinin (HA)-ubiquitin and increasing amounts of Myc-TIRM45 and treated with MG132 were immunoprecipitated with anti-Flag beads and immunoblotted with an anti-HA antibody. (b) U87 cells were transfected with plasmids expressing Flag-p53, Myc-TIRM45 or Myc-MDM2 together with HA-ubiquitin or its indicated mutants in the presence of MG132. The cell lysates were immunoprecipitated with anti-Flag beads and immunoblotted with an anti-HA antibody. (c and d) Lysates of U87 cells transfected with plasmids expressing Flag-p53, HA-tagged K63-linked ubiquitin (HA-K63-Ub) (c) or HA-K48-Ub, (d) together with Myc-TIRM45 or Myc-TIRM45 C29A and treated with MG132 were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibody. (e) Immunoblot analysis of extracts of U87 cells transfected with Flag-p53 and increasing amounts of HA-TRIM45 or HA-TRIM45 CA (f) Luciferase activity in U87 cells transfected with p53-luc, p53, and Myc-TIRM45 or Myc-TRIM45 C29A. Results are presented as Firefly luciferase activity relative to Renilla luciferase activity. Each bar represents the mean±S.D. of three independent experiments. **P<0.01. (g) A schematic diagram of p53 mutants. (h) Lysates of U87 cells transfected with plasmids expressing HA-K63-Ub and wild-type (WT) p53 or p53 mutant together with Myc-TRIM45 or empty vector and treated with MG132 were immunoprecipitated with anti-Flag beads and immunoblotted with an anti-HA antibody. (i) Immunoblot analysis of extracts of U87 cells transfected with plasmids expressing HA-K63-Ub and WT p53 or p53 mutant together with Myc-TRIM45 or empty vector