Figure 4
Effect of SKP2 overexpression on p27 protein levels and ubiquitylation (A) Asynchronous LNCaP cells were infected with adenovirus driving the expression of Myc epitope-tagged SKP2 (Ad-SKP2). Cell lysates were prepared at the indicated times after infection and Myc-SKP2 and p27 expression were determined by immunoblotting with p27 and Myc antibodies. Tubulin is shown as a loading control. (B) LNCaP cells kept in the presence of 10 ng/ml MIB for 72 h were infected with Ad-SKP2 or Ad-SKP2-ΔF (lacking the F-box) for increasing periods (24, 48, and 72 h in lanes 3–5 and 6–8, respectively). Protein lysates were prepared and assayed for the expression of the indicated proteins by immunoblotting. Overexpression of SKP2 is demonstrated by blotting with SKP2 (second panel from top) and Myc (third panel from top) antibodies. The first lane contains lysate from untreated controls. Tubulin is shown as loading control. (C) Effect of SKP2 on in vitro p27 ubiquitylation activity reconstituted in LNCaP cell lysate. LNCaP cells were arrested with 10 ng/ml MIB for 72 h and infected with Ad-SKP2 or Ad-SKP2-ΔF for 24 or 48 h as indicated. Cell lysate was prepared and employed in ubiquitylation assays using wildtype or T187A mutant p27 as substrate. Polyubiquitylated reaction products are indicated (p27-Ubn). The lower panel shows an immunoblot of the lysates used for the ubiquitylation assays downregulation of p27 by SKP2, but not SKP2-ΔF in these lysates.