E cadherin m 106

Uterine sections from paraffin-embedded tissue were cut at 6 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series. Sections were pre-incubated with 10% normal horse serum in PBS (pH 7.5) and then incubated with anti-Ki67 antibody (BD550609. BD Pharmingen) or anti-PTEN (9188, Cell Signaling), pAKT (4060, Cell Signaling), AKT (4691, Cell Signaling), ERK1/2 (4695, Cell Signaling), pERK1/2 (4370, Cell Signaling), Vimentin (ab92547, Abcam), E-cadherin (M-106, Takara), Cd133/Prominin (monoclonal 13A4, Invitrogen) in 10% normal serum in PBS (pH 7.5). On the following day, sections were washed in PBS and incubated with a secondary antibody (5 μl/ml; Vector Laboratories. Burlingame, CA USA) for 1 h at room temperature. Immunoreactivity was detected using the DAB kit (Vector Laboratories, Burlingame, CA USA).

Cells were lysed and sonicated for 10 sec on ice. Lysates were diluted to a protein concentration of 1 µg/µl and boiled for 5 min at 95°C. Equal amounts of proteins were separated on 8 and 10% gels and transferred to nitrocellulose membranes. Membranes were blocked [phosphate-buffered saline (PBS), 5% non-fat milk, 0.5% Tween] and immunostained with primary antibodies: E-cadherin M106 (Takara, The Netherlands), P-cadherin 610228 (BD Biosciences, Erembodegem, Belgium), vimentin V6389, α-catenin C2081, β-catenin C2206 and cytokeratin C2931, recognizing subtype (4, 5, 6, 8, 10, 13 and 18) (Sigma-Aldrich, St. Louis, MO, USA). Then, the secondary antibodies were applied, either ECL™ anti-mouse IgG or ECL™ anti-rabbit IgG (GE Healthcare UK Ltd., Buckinghamshire, UK). Immunodetection was performed with Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and imaged with ProXima 2850 (Isogen Life Science, De Meern, The Netherlands). HCT8/E11 was used as positive control for E-cadherin, P-cadherin and cytokeratin. MDA-MB-231 GFP Luc cells were used as a positive control for vimentin. Both cell lines were positive controls for α-catenin and β-catenin.