Mouse anti e cadherin antibody

Gastric cancer and control tissues were homogenized by grinding in liquid nitrogen and sonicated on ice in High KCl lysis buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 300 mM KCl, 1 mM EDTA, 0.5% Triton X-100 and 0.5% sodium deoxycholate) with complete protease inhibitor cocktail (Roche). Then the lysate was centrifuged at 13,000 g for 20 min at 4 °C. The supernatant was collected and quantified for protein concentration by BCA protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts of proteins were resolved by SDS-PAGE, and western blotting was performed with PVDF membrane transfer and detection with primary antibody incubation followed by secondary HRP-conjugated antibody incubation and ECL detection. The western blot gel image was obtained with an Minichemi 500 chemiluminescent imager (Sagecreation, Beijing, China). The antibodies that were used as follows: mouse anti-E-cadherin antibody (SantaCruz, 1:1000); rabbit anti-MPO antibody (PTG labs, 1:2000), mouse anti-GAPDH (PTG labs, 1:5000), goat anti-mouse HRP (PTG labs, 1:5000); goat anti-rabbit HRP (Signalway Antibody, College Park, MD, USA; 1:5000).

ICC cells (60% confluent) were deposited on coverslips and treated for 24 h with h-TGF-β1 with and without SB431542 or U0126 in 0.1% FBS media, then incubated with phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 2% sucrose, and permeabilized with 0.25% Triton X-100 followed by incubation with 2% BSA in PBS. Cells then were treated with mouse anti-E-cadherin antibody (Santa Cruz Biotechnology), followed by Alexa Fluor^® 488-conjugated anti-mouse IgG secondary antibody (Molecular Probes^®, Eugene, OR). Cell nuclei were stained with DAPI (Molecular Probes^®). Coverslips were mounted with Prolong^® gold antifade reagent (Molecular Probes^®) and examined under a confocal microscope (Olympus FV10; Olympus Corp., Tokyo, Japan).