Western blot analysis

TR Tejaswini P. Reddy
DC Dong S. Choi
AA Ann C. Anselme
WQ Wei Qian
WC Wen Chen
JL Johan Lantto
IH Ivan D. Horak
MK Michael Kragh
JC Jenny C. Chang
RR Roberto R. Rosato
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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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