Analysis of proteins was performed by Western blot as previously described [25]. Briefly, 30 μg protein of whole cell lysates were subjected to SDS-PAGE electrophoresis in 4 to 20% polyacrylamide gels (Bio-Rad, Hercules, CA, USA). Proteins were transferred onto nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with primary antibodies (1:1000), followed by washes and incubation with the appropriate secondary antibodies for 1 h (1:2000). Protein bands were developed in autoradiography films (Denville Scientific Inc., South Plainfield, NJ). Antibodies used in this study were purchased from Cell Signaling Technology and included anti-EGF Receptor (D38B1), anti-HER3/ErbB3 (D22C5), anti-phospho-EGFR (Tyr1068; D7A5), anti-phospho-HER3/ErbB3 (Tyr1289; D1B5), anti-phospho-FAK (Tyr925), anti-phospho-Stat3 (Tyr705), anti-Stat3 (79D7), anti-phospho-AKT (S4737), and anti-AKT (C67E7).
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