N cadherin n cad

Total protein was extracted by lysing cells in RIPA lysis buffer (KeyGene, Nanjing, China). After measurement of protein concentration, equal amounts of protein (30 μg per lane) were subjected to 10 % SDS-PAGE. After transferring onto the PVDF membranes (Bio-Rad), the membranes were blocked in 5 % skim milk (Beyotime), followed by immunoblotting with primary antibodies at 4℃for 10–14 h. Next, the corresponding secondary antibody (ab205718; 1:4000; Abcam, Cambridge, UK) was applied in combination with primary antibodies. Lastly, the combined signals were visualized with an enhanced chemiluminescence kit (KeyGene). In this study, the primary antibodies were purchased from Proteintech (Rosemont, IL, USA): E-cadherin (E-cad; 20874–1-AP; 2000), N-cadherin (N-cad; 22018–1-AP; 1:2000), GLUT1 (21829–1-AP; 1:2000), LDHA (21799–1-AP; 1:5000), ARF6 (20225–1-AP; 1:500) and β-actin (20536–1-AP; 1:2000).

With the application of RIPA lysis buffer (MedChemExpress, USA) and BCA Protein Assay Kit (Beyotime, Shanghai, China), the collection and quantification of total protein were successively performed. The protein samples were adjusted to the same amount (30 µg/lane) and subjected to 10% SDS-PAGE. The proteins were transferred to the membranes which were then immersed in 5% BSA and treated with primary antibodies against AKR1B10 (cat. no. ab192865; 1/1000; Abcam), HK2 (cat. no. ab209847; 1/1000; Abcam), matrix metallopeptidase 2 (MMP2; cat. no. ab181286; 1/1000; Abcam), matrix metallopeptidase 9 (MMP9; cat. no. ab76003; 1/1000; Abcam), Vimentin (cat. no. ab16700; 1/1000; Abcam), Slug (cat. no. ab302780; 1/1000; Abcam), pyruvate kinase M2 (PKM2; cat. no. ab85555; 1/1000; Abcam), lactate dehydrogenase A (LDHA; cat. no. ab52488; 1/5000; Abcam), E-cadherin (E-cad; cat. no. 20874-1-AP; 1/10000; Proteintech), N-cadherin (N-cad; cat. no. A0433; 1/1000; ABclonal) and β-actin (cat. no. ab8227; 1/5000; Abcam) at 4 °C overnight. Then, these membranes were incubated with HRP‐conjugated secondary antibody (cat. no. ab205718; 1/2000; Abcam) for 1 h. The bands were visualized using an ECL detection reagent (Millipore, Burlington, MA, USA). Densitometry analysis was conducted using ImageJ software (version 1.49; National Institutes of Health).

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.