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

Figure 7

Effect of BuA on the various isoforms. (a) EPN cells were treated with BuA 1 mM for 24 hours. Then, the nuclear extract was prepared and analyzed by bidimensional SDS PAGE and the isoforms were identified by MAb against [25, 26]. Form 0 corresponds to the wild type form and isoform 2 corresponds to the protein with 1 phosphate and isoform 4 corresponds to the CKI with 2 phosphate groups. Form 1 has not been identified and form 3 is the monophosphorylated derivative of form 1. Details on the identification are reported in [25]. Note that the immunoblotting reported corresponds to that which is showed in (b) (BuA-treated nuclear extract) but is exposed to the film for a long time period (i.e., 10 minutes). (b) EPN cells were treated or not for 24 hours with 1 mM BuA. Then, nuclear extracts were prepared and analyzed (1 mg extracts) by bidimensional analysis for p27 isoforms. (c) Exactly as in (b) except that cytosolic extracts were analyzed. (d) EPN cells were treated or not for 24 hours with 1 mM BuA. Then total extracts were prepared and immunoprecipitated with antibodies against phosphoserine 10- (pS10p27). The blot reports the input materials, the IP, the supernatant after pS10p27 removal, and an IP not related (NR). (e) EPN cells were treated for 24 hours with 1 mM BuA. Then cell extracts were prepared and divided into 2 equal aliquots. From one aliquot pS10p27 was removed by IP. Finally, the untreated aliquot and the aliquot from which pS10p27 was removed were analyzed by bidimensional immunoblotting.

(a)

(b)

(c)

(d)

(e)