Rabbit anti human e cadherin

Total protein was extracted from cells, and the concentration was determined using the BAC protein concentration assay kit (Beyotime Biotec, China). Samples were separated using 8–15% SDS-PAGE and then transferred to PVDF membranes (Millipore, NY, and the USA). Membranes were blocked for 1 h with 5% non-fat milk. Membranes were incubated with primary antibodies at 4°C. Antibodies used in this study were as follows: rabbit anti-human WNK3 (1:2,000; Abcam, UK), rabbit anti-human Cyclin D1 (1:2,000, Cell Signaling Tech), rabbit anti-human MMP-2 (1:2,000, Abcam), rabbit anti-human MMP-9 (1:2,000, Abcam), rabbit anti-human Snail1 (1:2,000, Abcam), rabbit anti-human E-cadherin (1:3,000, Abcam), rabbit anti-human Vimentin (1:3,000, Abcam), and mouse anti-human β-actin (1:2,000, Abcam). After three washes in TBST, the membranes were incubated with the appropriate secondary antibody (1:5,000, Abcam) in TBST for 2 h at room temperature. Proteins were detected using the ECL detection solution (Apexbio, Houston, USA).

CollagenaseΙA, trypsin, Melatonin and Matrigel were obtained from Sigma-Aldrich (St. Louis, MO, USA). Penicillin, DMEM/F12 (1:1) media were obtained from HyClone (Logan, Utah, USA). Charcoal-stripped fetal bovine serum (FBS) was obtained from GIBCO (Invitrogen, NY, USA). Rabbit anti-human E-Cadherin, N-Cadherin and Vimentin primary antibodies were obtained from Abcam (Cambridge, MA, USA). Rabbit anti-human Notch, Numb, Slug and Snail primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) for western blot and obtained from Abcam (Cambridge, MA, USA) for immunohistochemistry. Mouse anti-human β-Actin primary antibody and Goat anti-rabbit and Goat anti-mouse HRP-conjugated secondary antibodies were obtained from ZSGB-BIO (Beijing, China). Mammalian Cell Protein Extraction Kit was purchased from Beyotime (Shanghai, China). ECL Plus Western Blotting Detection System was obtained from Millipore Corporation (Billerica, MA, USA).

Cells were solubilized in radio-immunoprecipitation assay (RIPA, Beyotime) containing 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime) for 30 min on ice followed by centrifuging for 10 min at 4 °C, 12,000g. The supernatant was run on 12% sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime) and electrotransferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes for 1 h at 100 V. The PVDF membrane was probed with primary antibodies overnight at 4 °C. Antibodies used for western blot analysis were as follows: (1) rabbit anti-human Cyclin D1 (1:1000, Abcam, Cambridge, MA, USA), (2) rabbit anti-human cl-C3 (1:1000, CST, Danvers, MA, USA), (3) rabbit anti-human Twist (1:1000, Abcam), (4) rabbit anti-human E-cadherin (1:1000, Abcam), (5) rabbit anti-human Vimentin (1:1000, Abcam), (6) rabbit anti-human BRD4 (1:1000, Abcam), (7) mouse anti-human c-myc (1:1000, Abcam), (8) mouse anti-human BCL-2 (1:500, Santa Cruz, CA, USA). And then the membranes were incubated in 1:5000 HRP-labeled goat anti-rabbit IgG (CST) or horse anti-mouse IgG (CST). The proteins were visualized using the Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).