Histone Deacetylase Inhibitors Increase p27^Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

Figure 4

Effect of BuA on the proteasome degradation of . (a) EPN cells were treated for 8 hours with different inhibitors of proteolytic activities, that is, E64 (inhibitor of cysteine protease, 100 μM) and ALLN (N-acetyl-leucyl-leucyl-norleucinal, 50 μM) and CLBL (clasto-lactacystin β-lactone, 50 μM). After 8 hours of incubation, cells were collected and the contents of (p27) and Skp2 were determined by immunoblotting. Actin was analyzed for confirming equal loading. (b) On the left, EPN cells were treated with BuA for 24 hours or ALLN for 8 hours or BuA (24 hours) plus LLnL (the final 8 hours). Then, the abundance of (p27), Skp2, and actin was determined by immunoblotting. On the right, for the densitometric quantitation of analysis, three identical experiments were performed and the relative films were scanned and analyzed by ImageJ64 software. The data reported (as pixel) represent the mean values ± S.D. and compared to untreated cells (Con). (c) EPN cells were transfected for 24 hours with pcDNA3.0-ubiquitin-cMyc. Then, the cells were treated or not with BuA for 24 hours. Finally, cell extracts were analyzed by immunoblotting employing a monoclonal antiserum (MAb) directed against cMyc. On the right of blot are reported the molecular weight standard values (kDa). (d) K562 cells were treated (or not) with BuA for 24 hours or with ALLN or CLBL for 12 hours. Then, cellular extracts were prepared and analyzed for and actin content. The blots of were exposed for different times (as shown) in order to show the ubiquitination derivatives. On the top blot (right) are also reported the molecular weight standard values (kDa).

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