Anti egfr 1005 sc 03

Cells were lysed in lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1mM Na3VO4, 1mM NaF, 1% Triton X-100), or RIPA buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1mM Na3VO4, 1mM NaF, 0.1% SDS, 0.1% deochycholate, 1% IGEPAL CA-630) with protease inhibitor cocktail (Roche). Lysates were run on 8 or 10% SDS-PAGE and transferred to PVDF membrane (EMD Millipore). Proteins were detected using: anti-MET (D1C2 XP– Cell Signaling), anti-phospho-MET Tyr1234/Y1235 (Cell Signaling), anti-HER3 (SC-285 – Santa Cruz), anti-HER3 (SC-81455 – Santa Cruz), anti-HER3 (D22C5 XP – Cell Signaling), anti-phospho-HER3 Tyr1289 (21D3 – Cell Signaling), anti-HER2 (SC-284 – Santa Cruz), anti-phospho-HER2 Tyr1221/1222 (Cell Signaling), anti-FLAG M2 (Sigma-Aldrich), anti-EGFR ((1005) SC-03 – Santa Cruz), anti-phospho-EGF-Receptor Tyr1068 (Cell Signaling), β-tubulin (9F3 – Cell Signaling), GAPDH (D4C6R - Cell Signaling). Secondary antibodies were anti-rabbit-IgG HRP-linked antibody (Cell Signaling), or anti-mouse IgG HRP-linked whole antibody (GE Healthcare Biosciences). Blots were developed using ECL/ECL Prime (Thermo Fisher Scientific).

Dulbecco’s modified Eagle’s medium and penicillin-streptomycin solution were purchased from Mediatech (Herndon, VA). Cosmic calf serum was obtained from Hyclone (Logan, UT). Phosphate-buffered saline (PBS) (0.01 M phosphate; 0.138 M NaCl; 0.0027 M KCl; pH 7.4) and methanol was obtained from Sigma-Aldrich (St Louis, MO). Chloroform was purchased from Fisher Scientific (Pittsburgh, PA). Avidin-functionalized magnetic particles were obtained from Micromod (Rostock, Germany). Biotinylated anti-Epidermal Growth Factor Receptor (anti-EGFR) monoclonal antibody (111.6) was purchased from LifeSpan Biosciences (Seattle, WA). Biotinylated IgG1 isotype control was purchased from eBioscience (San Diego, CA). Anti-EGFR (1005, sc03) and horseradish peroxidase-conjugated secondary anti-rabbit IgG (sc-2054) were purchased from Santa Cruz Biotechnology (Dallas, TX). The nematic LC 4’-pentyl-4-cyanobiphenyl (5CB) was obtained from EMD Chemicals (Spring Valley, NY). Deionization of a distilled water source was performed with a Milli-Q system (Millipore, Bedford, MA) to give water with a resistivity of 18.2 MΩ-cm.