Ecl plus enhanced chemiluminescence reagents

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).

Cells were suspended in modified RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris–HCl [pH 7.4]), containing a protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitors (1 mM sodium fluoride and 2 mM sodium orthovanadate) on ice for 30 min, and centrifuged at 15,000 × g for 30 min to collect whole cell lysates. The proteins (10–20 μg) were separated on an 8%–12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Western blotting was performed with specific primary antibodies and peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies. Proteins were visualized with ECL Plus enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA). The commercial antibodies used in this study included HER2, pHER2, HSP90, AKT, pAKT, pERK, cPARP, BIM, and β-actin (Cell Signaling Technology, Danvers, MA, USA).