Phospho egfr

Cells were harvested and lysed in Nonidet P-40 buffer containing a protease-inhibitor mixture (Sigma). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on Mini-PROTEAN TGX Any kD Precast Gels (BioRad, Hercules, CA, USA) and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Immunoblots from tumor cell lysates were probed with antibodies against DDX3X (Sigma), EGFR, phospho-EGFR (Tyr1068), phospho-EGFR (Tyr1173), phospho-EGFR (Tyr845), Akt, phospho-Akt, Erk1/2, phospho-Erk1/2, and β-actin (Sigma). All antibodies except for anti-DDX3X and anti-β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Secondary antibodies consisted of anti-mouse IgG (BioRad) and anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, Cambridge, MA, USA). Immunoreactive protein bands were visualized using an ECL kit (Pierce). At least three independent experiments were performed for all analyses.

CHO cells were purchased from American Type Culture Collection (ATCC) and maintained in 10% CO₂ with Ham’s F-12K (Kaighn’s) medium (Gibco) supplemented with 10% FBS and 1% penicillin-streptomycin (both from Genessee Scientific). Antibodies recognizing the following proteins were purchased: Actin AC-15 (Sigma-Aldrich), EGFR, phospho-EGFR, phospho-EGFR Y1068, phospho-EGFR Y992, phospho-EGFR Y1045, Akt, phospho-Akt S473, p44/42 MAPK (Erk1/2), and phospho-Erk1/2 T202/Y204 (Cell Signaling Technologies). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Bio-Rad Laboratories. The primary antibodies, secondary antibodies, and dilutions are listed in Supplementary Table 16.