Ecl plus western blotting detection system

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).

Protein samples (60 μg) were heated to 95 °C for 5 min and loaded onto 10% sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% nonfat dry milk for 1 h at room temperature. Membranes were washed with 0.1% PBST (phosphate-buffered saline (PBS) and Tween20) three times and then incubated with primary antibodies overnight at 4 °C. Antibodies against pERK1/2 (Cell Signaling, Beverly, MA), STIM1 (Cell Signaling, Beverly, MA), and ORAI1 (GeneTex, Hsinchu, Taiwan) were diluted 1: 2000, whereas the antibody against β-actin was diluted 1: 10000. Membranes were then washed with 0.1% PBST three times and incubated with a 1: 5000 dilution of anti-mouse or anti-rabbit HRP-conjugated immunoglobulin G (IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After washing with 0.1% PBST three times, signals were detected by an ECL-plus Western blotting detection system (Millipore).