Both MDM2 and MDM2 (C464A) disrupt Axin-p53 interaction by recruiting p53.

<p>(A) HEK 293T cells were transfected with 2 µg untagged Axin and increasing amounts (3 µg and 8 µg) of HA-MDM2, HA-MDM2 (C464A) or HA-MDM2Δp53. (B) HEK 293T cells were transfected with HA-Axin, FLAG-p53 and Myc-MDM2 or MDM2 (C464A) and analyzed by immunoprecipitation and western blotting. (C) In normal lysis buffer, 1 µg purified His-Axin, 1 µg His-p53 and 6 µg GST-MDM2, GST-MDM2 (C464A) or GST-MDM2Δp53 were added and p53 was immunoprecipitated following by western blotting. BSA was used to make the protein amount of each sample equal. (D) HEK 293 cells were transfected with different combinations of pCMV5-LacZ, p53-luciferase reporter, HA-Axin, and Myc-MDM2 as indicated. At 6 h after transfection, cells were treated without or with 5 µM, 10 µM and 20 µM Nutlin 3a respectively for another 24 h followed by measure of luciferase activity. Results shown are mean±s.d. of three independent experiments. (E) U2OS cells were plated in 100 mm dishes at 70% confluence, followed by treatment with 0 µM, 0.4 µM and 2.5 µM doxorubicin respectively. 24 h after treatment, cells lysates were divided into two parts, one was immunoprecipitated with anti-Axin antibody, the other part was immunoprecipitated with anti-MDM2 antibody.