Western blot analysis of cell lines

Protein was retrieved at 70–80% confluent growth. After washing twice with PBS, cells were scratched from the plates, transferred into a 1.5 ml tube, and centrifuged for 5 min at 5000 rpm at 4 °C. The pellet was resuspended in M-Per buffer (Thermo Scientific) supplemented with cOmplete protease inhibitor mix (Roche) and Phosphostopp phosphatase inhibitor mix (Roche). After sonification for 5 min and centrifugation at 13000 rpm for 10 min at 4 °C, protein concentration in the supernatant was measured using the BCA Protein Assay Kit (Pierce) according to the manufacturer’s protocol. Twenty microgram of protein in 6x loading buffer (300 mM Tris, 600 mM DDT, 12% SDS, 60% glycerol, bromphenol blue according to desired intensity) was heated to 95 °C for 10 min, cooled on ice and loaded on 2% SDS stacking/10% SDS separation gels. PageRuler Prestained Protein Ladder (Thermo Scientific) was used as protein weight standard.

Electrophoresis was run at 100 V. Subsequently, proteins were transferred to nitrocellulose membranes by semi-dry blotting between filter papers soaked with blotting buffer (25 mM Tris, 150 mM glycine, 10% methanol). Nitrocellulose membranes were blocked with 5% milk powder in TBST for 30 min. Incubation with primary antibodies (Rabbit anti-β-catenin (#9562 Cell Signaling), Mouse anti-E-cadherin (#14472 Cell Signaling), Rabbit anti-β-tubulin (#2146 Cell Signaling), all 1:1000) was carried out overnight at 4 °C, incubation with secondary antibodies (horse anti-mouse HRP-linked IgG; 7076S or goat anti-rabbit HRP-lined IgG 7074S, both Cell Signaling), was carried out for 1 h at room temperature. Protein bands were detected using the ECL Western Blot Detection Reagent (Amersham) according to the manufacturer’s protocol. All incubation steps were followed by washing the nitrocellulose membranes with TBST three times. Antibodies were diluted in 5% milk powder in TBST.