Fig. 5. Mdm2 induces Tip60 degradation through their physical interaction. (A) Schematic representation of the Tip60 protein. The hatched box represents the chromodomain, the grey box the putative Zn finger and the black box the AcCoA binding site. Also indicated is the HAT domain, which is conserved among proteins of the MYST family. (B) U2OS cells were transfected with 10 µg of pCMV NeoBam Mdm2 and either 10 µg of pCDNA3 HA-Tip60 fl, pcDNA3 HA-Tip60 1–258 or pCDNA3 HA-Tip60 1–364. Whole-cell extracts were prepared 24 h after transfection, and subjected to immunoprecipitation with either the anti-HA antibody (lanes 2, 5 and 8) or an irrelevant antibody (anti-myc 9E10, lanes 3, 6 and 9), as indicated. Immunoprecipitates were tested for the presence of Mdm2 by western blotting. In lanes labelled inp (input), 10 µl of whole-cell extracts were loaded directly. In lanes 10–12, 10 µl of whole-cell extracts were subjected to an anti-HA western blot, to monitor the quantity of the various mutants in the extracts. Note that Tip60 1–258 is present in far larger amounts than Tip60 fl or 1–364, because these latter proteins are very poorly extractible from cells (compare with C). The asterisks indicate two non-specific bands. HA-Tip60 1–364 migrates just below a non-specific band. (C) U2OS cells were transfected as in Figure 1B with expression vectors of the Tip60 deletion mutant indicated (in pCDNA3-HA, 1 µg), pCMV 2N3T Ku80 (1 µg), pCMV luciferase reporter vector (100 ng), in the presence or absence of pCMV NeoBam Mdm2 (2 µg), as indicated. The amount of promoters in the transfection was kept constant using empty vectors. Luciferase activity was measured 24 h after transfection. The steady-state levels of HA-tagged proteins were assessed by western blotting.