Vanin-1 in Vascular Remodeling Disease Figure 6. Reduced neointima formation after carotid ligation in Vnn12/2 mice. A, Cross sections of representative H&E stained carotid arteries of Vnn12/2 and WT mice are shown, 21 days after carotid ligation. B–C, For each arterial section, intimal and medial areas were measured, and intima:media ratio calculated. Morphometric analysis was performed using NIH Image Analysis Software. Data are mean 6 SD of 3 independent experiments *P,0.05 WT vs. Vnn12/2. doi:10.1371/journal.pone.0039106.g006 460 nm) [40]. Cells were washed three times with PBS and lysed with potassium phosphate buffer (100 mM, pH 7.5) containing 0.1% Triton X-100 and 0.6% sulfosalicylic acid. Protein concentrations were determined by bicinchoninic acid (BCA) protein assay. Enzymatic assay was carried out using 5 mg of cell lysate in phosphate buffer (100 mM potassium phosphate buffer, pH 7.5) containing 2 mM pantothenate- AMC, 0.01% BSA, 0.5 mM DTT, 5% DMSO, and 0.0025% Brij-35 in a total volume of 100 ml reaction mixture. Reactions were carried at 37uC in the presence or absence of cell lysate and the fluorescence (Excitation 350 nm and emission 460 nm) was recorded every 2 min, with the change in fluorescence measured over a 30 min period. A standard curve was generated using purified recombi- nant vanin-1 (Abnova, Walnut, CA, catalog # H00008876-P01) under the same buffer conditions described above. Vanin-1 activity was normalized for total protein content. Pantetheinase activity was calculated by taking the slope at 30 min, fitting the data to the standard curve and normalizing for total protein content. Quantification of GSH To determine GSH and oxidized glutathione levels, we used an enzymatic recycling assay (glutathione assay kit, Cayman Chem- icals, Ann Arbor, MI) in the presence of glutathione reductase. We spectrophotometrically determined 5-thio-2-nitrobenzoic acid generation in deproteinated cells [38]. Measurement of Superoxide Generation Superoxide generation in SMCs was measured, as described [41], in cells incubated with dihydroethidium (DHE) (10 mM) in PBS at 37uC in the dark for 30 min in a 5% CO2 humidified chamber. Propidium iodide (10 mg/ml) was added 1 min before flow cytometry and examined by FACS with excitation at 488 and emission at 610 nm. Flow cytometry (FACScan; BD Biosciences, San Jose, CA) was used to select a homogeneous population of 10,000 live cells. Bivariate flow cytometry was performed with a FACS scan, and the data were analyzed with Cell Quest software (Becton Dickinson, San Jose, CA, USA), in the cell population from which apoptotic cells were gated out against forward and side scatter or PI-positivity. The geometrical mean of ethidium fluorescence intensity (excitation 488 and emission at 610 nm) in thepopulation was used for analysis. Alternatively, samples were examined by fluorescence microscopy (Advanced Microscopy Group, EVOS FL). Immunohistochemistry Formalin-fixed and paraffin-embedded tissue sections were deparaffinized and followed by rehydration, endogenous peroxi- dase activity was quenched using 3% H2O2. Followed by blocking, the sections were incubated overnight at 4uC with primary antibody against vanin-1, PPARc (1:100) and Ki-67 (1:100) as a cell proliferation marker, sections were then incubated with biotinylated secondary antibody and peroxidase-labeled PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39106