E cadherin n cadherin

For western blotting, cell lysates were first prepared using RIPA buffer (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s instructions. Protein samples were then applied to the wells of NuPAGE 4%–20% Tris-Gly gel, electrophoresed in sodium dodecyl sulfate running buffer (Invitrogen), and transferred to nitrocellulose membranes using the iBlot transfer apparatus (Invitrogen). Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) and 5% bovine serum albumin (BSA) for 1 hour at room temperature, followed by incubation with the primary antibody overnight at 4°C. On the following day, after the membranes were washed three times for 10 minutes each in TBS-T, horseradish peroxidase–conjugated secondary antibody (Bio-Rad, Hercules, CA) in TBS-T containing 2% BSA was applied for 1 hour at room temperature. Proteins were visualized with ECL Plus enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) using G-box Chemi Systems (SynGene, Bengaluru, India). The commercial antibodies used in this study were ERK, phospho-ERK, MEK, phospho-MEK, AKT, phospho-AKT, c-Raf, tetraspanin 7 (TSPAN7), E-cadherin N-cadherin, cleaved poly(ADP-ribose) polymerase (PARP), PARP, ITGB3, ITGAv, HSP90, Phospho-p90RSK, RSK, phospho-FAK and β-actin (all purchased from Cell Signaling Technology).