Anti egfr

Anti-human PTK6 (G6), anti-Raf-B (F-7) and anti-FAK (C-20) were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-EGFR and anti-P-PTK6 (Tyr-342) were purchased from Millipore (Temecula, CA). Anti-Ki67 was purchased from Abcam (Cambridge, MA). Antibodies directed against AKT, P-AKT (Thr-308), P-AKT (Ser-473), P-BCAR1 (Tyr-165), cleaved Caspase-3, P-EGFR (Tyr-845), P-FAK (Tyr-397), P-FAK (Tyr-576/577), p44/42 MAPK, p44/42 MAPK (Thr202/Tyr204) and P-FAK (Tyr-925) were purchased from Cell Signaling Technology (Danvers, MA). Anti-BCAR1 antibody was purchased from BD Biosciences (San Jose, CA). Antibodies directed against β-actin (AC-15) were purchased from Sigma-Aldrich (St. Louis, MO). Donkey anti-rabbit or sheep anti-mouse antibodies conjugated to horseradish peroxidase were used as secondary antibodies (GE Healthcare, Pittsburgh, PA) and detected by chemiluminescence with SuperSignal West Dura substrate from Thermo Fisher Scientific (Rockford, IL).

Total cellular lysates were prepared by solubilizing cells using cell lysis buffer containing 1% Triton X-100, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, and protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL). The lysates were mixed with Laemmli sample buffer for SDS-PAGE.
Nuclear fractions were prepared as described before (31 (link)), cells were washed with ice-cold PBS and scraped into Buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 2 mM MgCl₂, 1% NP40, and protease inhibitors), passed through a 21-gauge needle, and centrifuged. The pellet (nuclear fraction) was resuspended in Buffer B (20mM HEPES (pH 7.5), 10 mM KCl, 2 mM MgCl₂, 500 mM NaCl and 25% glycerol), homogenized, and kept on ice for 30 min. After centrifugation, the supernatant was mixed with Laemmli sample buffer for SDS-PAGE.
Western blot analysis was performed using anti-EGFR (Millipore, Billerica, MA), anti-phospho-EGFR (Tyr1068), anti-IκBα, anti-phospho-p38, anti-total-p38, anti-phospho-stress-activated protein kinase/c-Jun aminoterminal kinase (SAPK/JNK), anti-total-JNK, anti-total extracellular signal-regulated kinase (ERK)1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Promega, Madison, WI), anti-β-actin (Sigma-Aldrich), and anti-Ki67 antibodies.