Anti phospho smad3 c25a9

Following serum starvation for 24 h, subconfluent fibroblast cultures were either left untreated or stimulated with recombinant human TGF-β1 (rhTGF-β1) at a concentration of 5 ng ml -1 (R&D Systems, Minneapolis, MN, USA) for 60 min. Following this, cells were rinsed with ice-cold phosphate-buffered saline solution and then homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein lysates (35 or 40 μg) were separated on 10% sodium dodecyl sulfate-polyacrylmide gels and transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were probed with appropriate primary antibodies detected using peroxidase-conjugated antimouse or anti-rabbit antibodies and visualized by advanced chemiluminescence (Pierce, Rockford, IL, USA). The primary antibodies used were rabbit monoclonal anti-phospho-Smad3 (C25A9; Cell Signalling, Beverly, MA, USA) and rabbit polyclonal anti-p38 (Cell Signalling Technology, Berverly, MA, USA). The densities of the bands on the developed film were analyzed using UN-SCAN-IT gel software (version 6.1, Silk Scientific, Inc., Orem, UT, USA).