Human fibronectin

Analytical 12% SDS-PAGE was performed, and 30 μg of protein were analyzed for each condition, unless otherwise stated. For immunoblotting, proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane using an electroblot apparatus. Antibodies against human fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), FAK (Cell Signaling Technology, Danvers, MA), p-FAK (Epitomics, Burlingame, CA, USA), N-cadherin (Epitomics, Burlingame, CA, USA), E-cadherin (Epitomics, Burlingame, CA, USA), Snail1 (bs-1371R; Bioss, Boston, MA, USA), COX-2 (Lab Vision Corp., Fremont, CA), c-Jun (Santa Cruz Biotechnology), AKT and p-AKT (both from Cell Signaling Technology, Danvers, MA), p-Rac1/cdc42 (Cell Signaling Technology), and α-tubulin and β-actin (both from Sigma-Aldrich) were used as the primary antibodies. Mouse or rabbit IgG antibodies coupled to horseradish peroxidase were used as secondary antibodies. An enhanced chemiluminescence kit (Supersignal West Pico Chemiluminescence kit; Pierce, Rockford, IL) was used for detection. The FAK inhibitor Y15 was purchased from Sigma-Aldrich (St. Louis, MO).