The LC and PPS were homogenized in radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 5% SDS, 5% deoxycholic acid, 0.5 M Tris–HCl pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 200 mM sodium orthovanadate, and 200 mM sodium fluoride). Tissue extracts were incubated for 10 minutes on ice and clarified by centrifugation at 10,000g for 25 minutes at 4°C. Total protein in tissue extracts was measured using a standard bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Tissue extracts (30 μg total protein) were resuspended in 5× sample buffer (60 mM Tris–HCl pH 7.4, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue) at a 4:1 ratio, boiled for 5 minutes, and resolved by SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech, Germany), and blots were stained with Ponceau S (Sigma-Aldrich Corp., St. Louis, MO, USA) to visualize the protein bands and ensure equal protein loading and uniform transfer. Blots were washed and blocked for 45 minutes with 5% nondried skim milk in Tris-buffered saline with Tween-20 (TBST) buffer (20 mM Tris–HCl pH 7.6, 137 mM NaCl, and 0.1% Tween 20). Blots were then probed for 24 hours using antibodies against LOX, LOXL1, LOXL2, TGF-β, elastin, fribrillin-1, HDAC2, HDAC3, acetylated histone H3, and GAPDH (Sigma-Aldrich Corp.). Blots were then probed with horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody. Bound antibodies were detected using an enhanced chemiluminescence system (ECL, Amersham, MA, USA) and X-ray film. Relative intensity was measured using an ImageMaster VDS (Pharmacia Biotech, Sunnyvale, CA, USA) and the fold changes in these protein levels compared to GAPDH are indicated below the blot. Results are representative of five independent experiments. Data are expressed as mean ± SD.