Western Blot Analysis of Cellular Proteins

Cells were lysed with Laemmli sample buffer containing 2.5% β-mercaptoethanol and heated at 95°C for 5  min. Samples were loaded to 4–15% polyacrylamide gels (Bio-Rad) for electrophoresis. Proteins were then transferred to a Poly Vinylidene DiFluoride (PVDF) membrane (Merck), which was blocked with 5% dry milk, Tris buffered saline, 0.2% Tween, and incubated with primary antibodies (overnight at 4°C) followed by secondary antibodies (1:10000) for 1  hr at room temperature. Proteins were detected by using Luminata Crescendo (Merck) and LAS600 (GE Healthcare). The following antibodies were used: anti-MMP14 rabbit monoclonal (1:1000, EP1264Y, Abcam), anti-alpha-catenin rabbit monoclonal (1:1000, EP1793Y, Abcam), anti-Vimentin mouse monoclonal (1:1000, 1A4, Sigma), anti-Fibronectin rabbit polyclonal (1:1000, Sigma), anti-integrin β1 mouse monoclonal (1:1000, P5D2, Abcam), anti-integrin β3 rabbit monoclonal (1:1000, ERP17507, Abcam), and anti-actin mouse monoclonal antibody (1:2000, AC-40, Sigma).