Western Blot Analysis

Total proteins from OS cells were extracted in RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM Na₃VO₄) and quantified using the Bradford method. Protein samples (30 μg) were separated by SDS/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using wet transfer. After blocking non-specific antibody binding sites, the membrane was incubated overnight at 4°C with mouse monoclonal antibodies against the indicated antibodies. Vimentin (sc-6260), Twist (sc-81417), VEGF (sc-507), and MMP-2 (sc-10736) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). And N-cadherin (4061s), Snail (3895s), and β-actin (3700) were obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody was purchased from Invitrogen (Carlsbad, CA). After incubation with peroxidase-conjugated secondary antibodies at 37°C for 1 h, the protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, PA) and detected using the Amersham Imager 680 (GE Healthcare Biosciences).