PPARα inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16^INK4a
J. Clin. Invest. Florence Gizard, et al. 115:3228 doi:10.1172/JCI22756 [Go to this article.]

Figure 3
PPARα activation increases p16 protein levels and p16-CDK4 interaction and decreases CDK4-mediated pRB phosphorylation in hSMCs and mSMCs. (A–C) Protein extracts of hSMCs infected and treated for 12 hours (A) or 24 hours (B and C) with GW7647 (600 nM) were submitted to Western blot analysis either directly (A and B) or after IP with an anti-CDK4 antibody (C). Cellular p16 protein levels (A) and in-cell CDK4-mediated pRB phosphorylation (B) were measured using an antibody specific for p16 or an anti-pRB antibody recognizing a site specifically phosphorylated by cyclin D1/CDK4 (P∼Ser780-ppRB) (28), respectively. (C) Anti-CDK4 immunoprecipitates were assayed for kinase activity using the 769–921 pRB fragment as substrate (63) (top panel), or analyzed by Western blot using specific anti-p16 (middle panel) or anti-CDK4 antibodies (lower panel). (D) Protein extracts of PPARα^–/– or PPARα^+/+ C57BL/6J mSMCs treated for 24 hours with Wy14,643 (6 μM) were submitted to Western blot analysis using the anti-p16 and anti–P∼Ser780-ppRB antibodies.