Phospho egf receptor tyr1068

For immunoblot analysis, tumor tissues and human pancreatic cells (BxPC-3, Panc-1, and MIA PaCa-2) were homogenized or lysed, respectively, with ice-cold RIPA buffer (Pierce; Thermo Fisher Scientific, Inc.) supplemented by a protease and phosphatase inhibitor cocktail (Pierce; Thermo Fisher Scientific, Inc.) in agreement with the manufacturer's procedures. The protein fractions were calculated using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Tissues were homogenized and cell lysates were subjected to SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and then transferred to a sheet of nitrocellulose membranes (Macherey-Nagel, Germany). The blots were blocked with 5% nonfat dried milk and incubated with primary antibodies Cleaved Caspase-3 (#9661), Bcl-xL (#2764), Survivin (#2808), EGF Receptor (D38B1) XP (#4267), Phospho-EGF Receptor (Tyr1068) (#2234), Akt (#4685), Phospho-Akt (Ser473) (#4060), (Cell Signaling Technology, Beverly, MA) and actin, clone C4 MAB1501 (Millipore, Billerica, MA) overnight, at 4°C. Next, membranes were then re-probed with secondary antibodies for 1h at room temperature, followed by incubation with ECL reagent (Amersham Biosciences), and finally exposed to X-ray film (Fuji, Fischer Scientific). The ImageJ platform (NIH) was employed for protein quantification.

The primary antibodies MAPK (Erk) (#9102, 1:1000), Akt (1:1000, #9272), Phospho-EGF Receptor (Tyr1068) (1:1000, #2234), p(Thr308)-Akt (1:1000, #9275), Phospho-Akt (Ser473) (1:1000, #9271), Phospho-MAPK (pERK) (1:1000, #9101), β-Actin (1:10000, #4967) were purchased from Cell Signaling (Leiden, The Netherlands) and anti-EGFR (1:1000, sc-03-G) was purchased from Santa Cruz Biotechnology(Texas, USA). Cetuximab (ERBITUX) was ordered from Merck (Dietikon, Switzerland). Gefitinib (Iressa) was bought from Sigma (Zwijndrecht, The Netherlands); sunitinib was purchased from LC Laboratories (Woburn, USA). Entinostat and SAHA were purchased from Selleckchem (Munich, Germany). Staurosporine and cisplatin were purchased from Sigma-Aldrich (Zwijndrecht, Nederland). Doxorubicin was purchased from Teva Pharmaceuticals. All drugs were aliquoted in DMSO and stored at -20°C. The human epidermal growth factor (hEGF) and platelet-derived growth factor (PDGF) were purchased from Sigma-Aldrich (Zwijndrecht, Nederland).