Anti γ catenin

Total protein was extracted from the collected cells. Cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Membranes were subsequently blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk followed by incubation with primary antibodies overnight at 4°C. After a second incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Multi Sciences Biotech, Hangzhou, China), the bands on the PVDF membranes were developed using enhanced chemiluminescence (ECL; Biological Industries, Beit HaEmek, Israel). All primary antibodies (anti-γ-catenin, anti-β-catenin, anti-Cyclin D1, anti-c-Myc, anti-GSK3β, anti-phospho-GSK3β, anti-Histone H1 and anti-β-actin) were obtained from Cell Signaling Technology (Beverly, MA, USA).

Cells were lysed in RIPA buffer and analyzed by Western blotting as described (Huang et al. 2014 ). Antibodies used for Western blotting were as follows: anti-CD44 (R&D Systems), anti-hnRNPF (Santa Cruz Biotechnology), anti-E-cadherin (Cell Signaling Technology), anti-γ-catenin (Cell Signaling Technology), and anti-vimentin (NeoMarkers). GAPDH (GE) was used as a loading control. For immunofluorescence, cells were fixed in 4% polyformaldehyde and permeabilized with 0.2% Triton X-100 before blocking with 5% BSA. Primary antibody incubations with rabbit anti-E-cadherin (1:200; Cell Signaling Technology) were performed overnight at 4°C followed by incubation with Alexa fluor 488-conjugated anti-rabbit IgG (1:500; Invitrogen) for 2 h at room temperature and DAPI staining. Representative images were captured under a Nikon C2 laser scanning confocal microscope.