T egfr

Protein aliquots of 25 μg each were resolved by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA) or 1000 μg aliquots of total proteins were immunoprecipitated with the appropriate antibodies. The immune complexes were recovered with Protein G-Sepharose or Protein A-Sepharose beads (Zymed Laboratories, California). Electrophoresed protein samples or immunoprecipitated samples were transferred to polyvinylidene difluoride membranes (Bio-Rad). After washing three times, the membranes were incubated with blotting-grade blocker (Bio-Rad) for 1 h at room temperature and overnight at 4 °C with primary antibodies to p-AXL (Tyr702), t-AXL, p-EGFR, p-IGF-1R, t-IGF-1R, p-Akt (Ser473), t-Akt, p-Gab1, t-Gab-1, p-Shc, t-Shc, IRS-1,β-actin (13E5) (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), p-Erk1/2 (Thr202/Tyr204), t-Erk1/2, and t-EGFR (1:1000 dilution, R&D systems).
After washing three times, the membranes were incubated for 1 h at room temperature with HRP-conjugated species-specific secondary antibody. Immunoreactive bands were visualized using SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce Biotechnology). Each experiment was independently performed at least three times.

The following primary antibodies were used for indicated experiments; western blotting: p-ALK (Tyr1604), t-ALK, p-HER3, t-HER3, β-actin (13E5), t-HER2, p-Akt, t-Akt, E-cadherin, Vimentin, TCF8/ZEB1, and GAPDH, (1:1000 dilution; Cell Signaling Technology), and p-Erk1/2 (Thr202/Tyr204), t-ERK1/2, and t-EGFR (1:1000 dilution; R&D systems); immunohistochemistry: Vimentin (ACR 048A, C; Biocare Medical, Concord, CA, USA), E-cadherin (M3612; Dako, Santa Clara, CA, USA) and ZEB1 (ab180905; Abcam, Cambridge, UK).