FIGURE 4.

EBP50 regulates Skp2 stability and VSMC proliferation in an Akt-dependent manner. A, primary VSMC from EBP50^−/− mice, were electroporated with control empty vector (pcDNA3), WT, or [T156A]EBP50 and treated with cycloheximide (10 μg/ml) for the indicated times. Equal amounts of proteins were analyzed by Western blot for Skp2 expression. Data show the mean (± S.E.) of Skp2 intensity (normalized by β-actin) relative to time 0. *, p < 0.05, n = 3. B, VSMC transfected as in A, were treated with cycloheximide (10 μg/ml) in the absence or presence of the proteasome inhibitor MG132 (2 μm) for 2 h, followed by immunoblotting for Skp2 and Flag expression. C, VSMC transfected with WT and [T156A]EBP50 were treated with control siRNA or siSkp2. Proliferation was determined by [³H]thymidine incorporation. *, p < 0.05 versus vector, n = 3. D, Skp2 overexpression decreases p21^cip1 and p27^kip1 expression. Primary VSMC were electroporated with WT or [L424A]Skp2 and analyzed for the expression of p21^cip1 and p27^kip1 by Western blot. E, proliferation of VSMC overexpressing WT or [L424A]Skp2. *, p < 0.05 versus vector, n = 3.