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

Figure 6

Mechanisms of BuA activity on Skp2 abundance. (a) K562 cells were incubated for 16 hours with 1 mM BuA. Then, the nuclear and cytosolic compartments were prepared. Finally, content of Skp2 was determined by immunoblotting. Histone H3 and β-tubulin were used as markers of nuclear and cytosolic fraction, respectively, and for equal loading evaluation. (b) EPN cells were transfected with three different convalidated siRNAs directed against Skp2 mRNA. After 72 hours, the amount of Skp2, , and actin content was determined by immunoblotting. In the brackets the catalog number of the siRNA is reported. (c) EPN cells were transfected with pcDNA3-Skp2 plasmid or treated with BuA (or both). After 48 hours, the amount of Skp2 and (p27) content was determined by immunoblotting. Actin was evaluated for confirming equal loading. (d) K562 cells were cultured with or without BuA for 16 hours. Then, cell extracts were prepared and analyzed by immunoblotting for GSK3β and phospho-GSK3β (pGSK3β). Actin was also determined for confirming equal loading. (e) K562 cells were cultured with or without BuA for 16 hours. Then, cell extracts were prepared. CDK2 was immunoprecipitated from control (Con, untreated) and treated cells. On the top is showed CDK2 abundance in the input extracts, CDK2 remaining in the supernatants (to confirm the total precipitation), and CDK2 in 1/10 of IP. An IP not related (NR) is also showed. On the bottom is reported the mixture of assay. CDK2 immunoprecipitated from Con and BuA was employed to phosphorylate recombinant on threonine 187. The mixture of assay was analyzed for the content of and phosphothreonine 187- (pT187p27). The IP not related was also employed as control of phosphorylation.

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