Figure 3. Skp2 cooperates with Myc to regulate RhoA transcription independent of Myc ubiquitination and SCF-Skp2 E3 ligase activity.
(a, b) RhoA reporter assay in 293T cells transfected with the indicated plasmids. The number indicates the amount (μg) of the plasmids used in this assay (lower panel). Western blot analysis showing the expression of constructs as indicated (upper panel). (c) ChIP assay in PC-3 cells with GFP or Myc knockdown. The quantified results were presented as mean ± s.d. (n=3) (d) RhoA reporter assay in 293T cells transfected with various RhoA promoter constructs with mutations in different E-box regions. (e) RhoA reporter assay in 293T cells with GFP or Skp2 knockdown (left panel). Western blot analysis of Skp2 expression in 293T cells with GFP or Skp2 knockdown (right panel). (f) Analysis of RhoA and Skp2 mRNA levels in Wt and Skp2−/− primary MEFs by real-time PCR. The quantified results were presented as mean ± s.d. (n=3). (g) Western blot analysis of active RhoA (RhoA-GTP) and total RhoA expression in Wt and Skp2−/− primary MEFs. (h) Western blot analysis of RhoA expression in various cancer cell lines infected with GFP shRNA, Skp2 shRNA. Two Skp2 lentiviral shRNAs were used in this assay. (i) Western blot analysis in PC-3 cells infected with pBabe or pBabe-Skp2. Results in panel a, b, d, e were shown as mean ± s.d. of one representative experiment (from 3 independent experiments) performed in triplicate.