1/2, ERK 1/2, p-AKT, and AKT as we previous described [39]. The membrane was reprobed with secondary antibody for 1 h. Blots were processed using an ECL kit (Amersham Biosciences) and exposed to the film. Measurement of mineralized matrix formation in cultured CVSMCs Measurment was determined as we previously described [38]. CVSMCs in 24-well plates were cultured in DMEM containing 10% FBS in the presence of 10 nM insulin or vehicle for 12 days. The extent of mineralized matrix was determined by Alizarin Red S staining. Briefly, cells were fixed in 70% ethanol for 1 h at room temperature and stained with 40 mM Alizarin Red S for 10 min. Next, cell preparations were washed with PBS to eliminate nonspecific staining. The stained matrix was photographed using a digital microscope. Alizarin Red S staining was released from cell matrix by incubation in cetyl-pyridinium chloride for 15 min. The amount of released dye was quantified by spectrophotometry at 540 nm. 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(2010) PKA-induced receptor activator of NF-kappaB ligand (RANKL) expression in vascular cells mediates osteoclastogenesis but not matrix calcification. J Biol Chem 285: 29925–29931. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29037