Rabbit monoclonal anti n cadherin

The following antibodies were used: rabbit monoclonal anti-Orai1 and mouse monoclonal anti-vinculin (Abcam, Cambridge, UK); rabbit polyclonal anti-Pyk2 and mouse monoclonal anti-E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-phosphorylated Pyk2 (p-Pyk2) (Tyr402; R&D Systems, Minneapolis, MN, USA); rabbit monoclonal anti-N-cadherin and rabbit monoclonal anti-vimentin (Cell Signaling Technology, Danvers, MA, USA); Alexa Fluor 594-conjugated goat anti-mouse IgG (H + L) antibody (Invitrogen, Carlsbad, CA, USA); Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) antibody (Cell Signaling Technology).
Important reagents were as follows: thapsigargin, SKF96365, puromycin, and G418 from Sigma-Aldrich (St. Louis, MO, USA); and Fluo-4/AM and Pluronic-127 from Invitrogen. Boyden chambers were purchased from Millipore (Billerica, MA, USA) and Matrigel was purchased from BD Biosciences (San Jose, CA, USA). Confocal Petri dishes were obtained from NEST Biotechnology (Wuxi, JS, China).

Total protein was isolated from cultured cells using cOmplete Lysis-M EDTA-free and cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland). Protein concentrations were assessed using a BCA protein assay kit (Thermo Fisher Scientific). Equivalent amounts of protein samples were mixed with NuPAGE LDS Sample Buffer (4×), separated in NuPAGE Novex 4–12% Bis-Tris Gels (Life Technologies), and transferred onto nitrocellulose membranes. The membranes were blocked with blocking buffer (TBS-T [25 mM Tris-HCl pH 7.4, 125 mM NaCl, 0.05% Tween-20] with 5% bovine serum albumin [Sigma-Aldrich, St. Louis, MO, USA]) for 1 h, and incubated overnight at 4°C with the following primary antibodies and subsequently with corresponding secondary antibodies for 1 h at room temperature. Antibodies and dilutions used were rabbit monoclonal anti-E-cadherin (1:1000, Cell Signaling Technology, Danvers, MA, USA), goat polyclonal anti-Snail (1:1,000; Abcam, Cambridge, UK), rabbit monoclonal anti-N-cadherin (1:1,000; Cell Signaling Technology), rabbit monoclonal anti-vimentin (1:1,000; Cell Signaling Technology), and mouse monoclonal anti-β-actin (1:5,000; Santa Cruz Biotechnology, Dallas, TX, USA). Immunoreactive bands were detected using the ChemiDoc XRS + Imaging System (Bio-Rad Laboratories), and protein levels are presented relative to that of β-actin.