Protein extraction and Western blot

Tumor cell xenografts were cut into 1 × 1 mm pieces on ice and grinded and then scraped into 1 mL of the lysis buffer containing 1% Triton-100, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM pepstatin, 0.5 mg/mL, leupeptin, 0.3 μM aprotinin, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl. After being lysed on ice for 30 min, the protein samples were centrifuged at 15,000 rpm for 20 min, the supernatants transferred into new tubes, and protein concentration was assayed using the BCA protein assay kit (CWBIO, Beijing, China). For Western blotting, these protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10–15% of SDS-PAGE gels and transferred onto polyvinyldine diflouride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% bovine serum albumin (BSA) and incubated for 18 h at 4°C with a monoclonal anti-EGFR, anti-phospho-EGFR, anti-MAPK, anti-phospho-MAPK, anti-Akt, anti-phospho-Akt, or anti-β-actin antibody (CWBIO). The next day, the membranes were washed for three times with Tris-based saline-0.1% Tween 20 (TBS-T) and then further incubated for 2 h at room temperature with an HRP-conjugated secondary antibody (CWBIO). Immunoreactive protein bands were further visualized by using the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA) and exposed to X-ray films.