Anti fibronectin clone 10

10⁴ cells were seeded onto an 8-well Lab-TekII chamber slide, fixed in 3.7% formaldehyde solution (Sigma), and permeabilized in 0.1% Triton 100X (Sigma), PBS buffer at room temperature for 10 min. The cells were then washed 3 times with PBS and incubated for 1 h with 10% horse serum in PBS blocking solution. The cells were incubated overnight at 4°C with murine monoclonal anti-E-cadherin clone 36, (Becton Dickinson), anti-β-catenin clone 14 (Becton Dickinson), anti-fibronectin clone 10 (Becton Dickinson), anti-vimentin clone V9 (Dako), or with a polyclonal rabbit anti-HA Y11 (Santa-Cruz) primary antibody, washed in PBS, and then incubated for 1 h at room temperature with a goat anti-mouse Alexa Fluor 533 secondary antibody (A21422, Invitrogen) or with a mouse anti-rabbit Alexa Fluor 555 secondary antidody (A21428, Invitrogen). After extensive washes in PBS, the nuclei were stained with 5 mg/ml Hoechst for 10 min and mounted with Fluoromount-G (SouthernBiotech). All matched samples were photographed with an immunofluorescence microscope (Leica) and identical exposure times.

Cells were washed twice with a PBS- 0.5% EGTA solution and lysed in RIPA buffer (150 mM NaCl, 1% NP40, 100 mM Tris pH 7.5, 0.1% SDS, 0.5% DOC, 1 mM EGTA) supplemented with complete protease (Roche) and phosphatase inhibitors (Sigma). After clarification by centrifugation at 14 000 rpm for 20 min at 4°C, cell extracts were subjected to SDS-PAGE. Proteins were revealed with mouse monoclonal anti-E-cadherin (clone 36, Becton Dickinson), anti-β-catenin (clone 14, Becton Dickinson), anti-fibronectin (clone 10, Becton Dickinson), anti-vimentin (clone V9, Dako), anti-N-cadherin (Becton Dickinson), anti-phospho-ERK1/2 (9106S, Cell Signaling), anti-GAPDH (6C5, Biodesign), rabbit monoclonal anti-active caspase 3 (ab32042, Abcam), or rabbit polyclonal anti-SNAIL1 (ab17732, Abcam), anti-SNAIL2 (G-18/SC-10436, Tebu-bio), anti-SNAIL3 (HPA016757, Sigma), anti-ERK1/2 (#9102, Cell Signaling), anti-AKT (8272, Cell Signaling), anti-phospho-AKT (Ser473) (#4058, Cell Signaling), anti-c-MYC A14 (sc-789, Santa Cruz Biotechnology), anti-HA Y11 (Santa-Cruz) and horseradish-peroxidase-conjugated secondary antibodies (Dako). Antigen-antibody complexes were revealed with a reagent for western blotting (Santa Cruz).