Anti ddx3x

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.