Western blot analysis and phosphoinositide 3-kinase (PI3-kinase) inhibition

Proteins were isolated from cultured VSMCs, and the protein levels were determined by Western blot analysis. Briefly, equal amounts of protein were subjected to SDS-PAGE. Standard Western blot analysis was conducted using antibodies for Akt (1:1000 dilution; Cell Signaling, Danvers, MA, USA), phosphor-Akt (p-Akt, ser473, 1:1000 dilution; Cell Signaling) and KLF5 antibodies (1:1000 dilution; Abcam, Cambridge, MA, USA). GAPDH antibody (1:5000 dilution; Cell Signaling, Danvers, MA, USA) was used as a loading control. In cultured VSMCs, PI3-kinase/Akt pathway was inhibited by its inhibitor, LY294002 (20 μmol/L, Sigma-Aldrich, St Louis, MO, USA).