Fig. 2. Tip60 is subjected to proteasome-mediated degradation. (A) Jurkat cells (left panel) or HeLa cells (right panel) were treated or not with the proteasome inhibitor indicated (Lact., lactacystine). Total cell extracts were then tested for the presence of Tip60 by western blotting using either the anti-Tip60 DT antibody (left panel) or the anti-Tip60 SK antibody (right panel). The band indicated Tip60 co-migrates with the exogenous HA-Tip60 produced from pCDNA3 (data not shown). The asterisk (*) indicates a non-specific band recognized by the anti-Tip60 DT antibody. (B) U2OS cells were transfected as in Figure 1B. Twenty-four hours after transfection, the proteasome inhibitor ALLN was added where indicated (lanes 5–8). After 2 h, cycloheximide was added and cells were harvested after treatment for the length of time indicated. Total cell extracts were analysed as described in Figure 1A. (C) U2OS cells were transfected with 10 µg of pCMV 2N3T Tip60 and 10 µg of either pSG5 His-ubiquitin (lane 2) or pSG5 HA-ubiquitin (lane 1). Twenty-four hours after transfection, cells were lysed as described in Materials and methods and His-tagged proteins were purified by nickel chromatography. Nickel binding proteins were subjected to a western blot using the anti-HA antibody. The arrow points to the unmodified HA-Tip60 protein that harboured background binding to nickel beads.