Pegfr1045

The PC cells were seeded into 12-well plates with 1×10⁵ cells/ml with or without EGF (50 ng/ml). and cultured 48 h after transfection. Total proteins were extracted from cells or tissues with RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM PMSF and phosphatase inhibitor (Bimake, USA). The total protein concentration was determined by BCA Protein Assay Kit (TaKaRa, Japan). Proteins were separated on a 10% SDS–PAGE and then transferred onto PDVF membranes. Subsequently, membranes were blocked with 5% skimmed milk and incubated with primary antibodies: EGFR (Proteintech), the phosphorylated EGFR at tyrosine 1045 (pEGFR1045, Cell signaling technology) and at tyrosine 1068 (pEGFR1068, Abcam), E-cadherin (Abcam), c-Myc (Proteintech), ERK (Cell signaling technology), p-ERK (Cell signaling technology), MMP9 (Proteintech), mTOR (bimake), the phosphorylated mTOR at Ser2448 [p-mTOR (2448), Cell signaling technology], Akt (Proteintech), the phosphorylated Akt at Ser473 [p-Akt (ser473), Cell signaling technology], GAPDH (Proteintech). On the following day, membranes were incubated with horseradish-peroxidase-conjugated secondary antibodies (Proteintech). Protein bands were detected with an ECL detection kit (Thermol Biotech Inc, USA).

Whole-cell lysates were prepared from PC specimens and cell lines. Cells were pretreated with 10 ng/ml of TGF-β1 (Peprotech, RockyHill, New Jersey, USA) plus Notch signaling inhibitor RO4929097 (10 μM, Selleckchem, USA) for 24 h to detect the activity of TGF-β1-Smad2/3-Snail signaling. All samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes (Millipore Corp, Bedford, MA, USA), and incubated with primary Numb (Abcam,1:1000), ZEB-1 (Proteintech, 1:1000), Fibronectin (Proteintech, 1:1000), E-cadherin (E-cad, Abcam, dilution: 1:1000), N-cadherin (N-cad, Proteintech, 1:1000), Vimentin (Proteintech, dilution: 1:1000), Smad2/3 (Cell Signaling Technology, Beverly, USA, dilution: 1:500), p-ERK (Cell Signaling Technology, dilution: 1:1000), Snail1 (Proteintech, 1:500), Snail2 (Proteintech, 1:500), cleaved-Notch1 (Cell Signaling Technology, dilution: 1:1000), phosphorylating EGFR at tyrosine 1045 (p-EGFR1045, Cell Signaling Technology, dilution: 1:1000) and GAPDH (Proteintech, 1:3000) overnight at 4 °C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Proteintech) for 2 h at room temperature. Immunoreactive protein bands were visualized with an ECL detection kit (Thermol Biotech Inc, USA). Each experiment was repeated three times.