Active nonphosphorylated β catenin

Immunocytochemistry was performed using antibodies to EMT markers that included E-cadherin and vimentin (Millipore, Burlington, MA, USA), N-cadherin (R&D Systems, Minneapolis, MN, USA), metalloproteinase 9 (MMP9) and P120 (Abcam, Cambridge, MA, USA), and active (non-phosphorylated) β-catenin (Cell Signaling, Danvers, MA, USA). Also, an early endosome antigen 1 (EEA1) antibody (Cell Signaling, Danvers, MA, USA) was used for the E-cadherin colocalization study. Briefly, cells were grown in Ibidi chamber slides (Ibidi, Munich, Germany) and fixed using 4% paraformaldehyde. Cells were blocked in 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) in 0.1% Triton-X (Bio-Rad, Hercules, CA, USA) in PBS, and primary antibodies were diluted in 0.2% Tween (Sigma-Aldrich, St. Louis, MO, USA) in PBS and incubated at 4°C overnight. Cells were next washed in PBS then incubated in Alexa Fluor 594 and 488 secondary antibodies (Life Technologies, Eugene OR USA) at room temperature for 1 hour, then mounted in Vectashield (Vector Laboratories, Burlingame, CA USA).