Erbb2

Western blotting was performed as previously described.^{19 (link),30 (link)} Cell lysates were collected, and the protein concentration was determined by the Bradford assay (Bio-Rad, CA). The protein samples were electrophoresed on SDS-PAGE gels, transferred to PVDF membranes, and analyzed using antibodies against α-tubulin, phospho-AKT (S473), total-AKT, phospho-ERK (Thr202/Tyr204), total-ERK, phospho-STAT3 (Tyr705), STAT3, p21, SMARCA5 (Santa Cruz Biotechnology, USA), SMRT/NCOR2 (Santa Cruz Biotechnology, USA), ErbB3 (Santa Cruz Biotechnology, USA), ErbB2 (Santa Cruz Biotechnology, USA), human nestin (Santa Cruz Biotechnology, USA), and L1CAM (Fisher, USA). All antibodies were purchased from Cell Signaling, USA, unless otherwise indicated.

The cells were harvested in lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5% Triton X-100, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadate, 20 mM sodium fluoride, and 100 mM phenylmethylsulfonyl fluoride) containing Xpert protease inhibitor cocktail solution (genDEPOT Inc., Barker, TX) and incubated on ice for 50 min. Proteins (30 μg) were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 hour at room temperature and incubated with the appropriate primary antibodies at 4°C overnight, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized using the ECL system (GE Healthcare, Piscataway, NJ), and the images were developed on X-ray film (Agfa HealthCare NV, Mortsel, Belgium). Antibodies for the estrogen receptor, ErbB2, p53, Bcl-2, and Bax were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies for CAV-1, poly-ADP-ribose polymerase, and pBcl-2 were purchased from Cell Signaling Technology Inc. (Danvers, MA). β-Actin was obtained from Sigma-Aldrich Co. LLC (St. Louis, MO).