Western Blot Analysis of Biomarkers

A standard Western blotting protocol was used as described previously [5 (link),25 (link)] with antibodies against TGFβ (1:600), matrix metalloproteinase 2 (MMP2, 1:200) (Santa Cruz, CA, USA); connective tissue growth factor (CTGF, 1:500) (Biovision, Mountain View, CA, USA), LOX-1 (1:1000) (R&D System), bone morphogenetic protein-7 (BMP-7, 1:5000) (AbDSerotec, Minneapolis, MN, USA), nuclear factor-kappa B (NFκB, 1:1000), its inhibitor kappa B alpha (IκBα, 1:200), Phospho-P38 (1:1000) (Millipore, Billerica, MA, USA), NADP(H) oxidase subunit Gp91phox (1:500, BD Transduction Laboratories, France). Loading controls were β actin (1:3000, Sigma Aldrich, France) or P38 (1:1000, Millipore). Appropriate HRP-coupled secondary antibodies (1:5000 to 1:10 000, GE Healthcare, France) were used to detect the band by chemiluminescence with ECL plus (GE Healthcare, France). Intensities of the protein bands were determined and quantified using AlphaEase FC software (Alpha Innotech Corporation, San Leandro, CA).