Western Blot Analysis of E-cadherin

Cells were washed twice to exclude dead cells. Then, cells were lysed using extraction buffer (Life Technologies, Carlsbad, CA, USA), supplemented with phenylmethanesulfonylfluoride (0.1 mM PMSF) (Life Technologies, Carlsbad, CA, USA) and a protease inhibitor cocktail (1 μg/mL aprotinin, 1 μg/mL leupeptin) (Life Technologies, Carlsbad, CA, USA), according to manufacturer’s instructions. Cells were then centrifuged and supernatant was collected and submitted to protein quantification by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Waltham, MA USA). SDS-Page and Western Blot were performed, as described in [54 (link)]. Membranes were incubated with rabbit anti- E-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-GAPDH (GeneTex, Irvine, CA, USA), and goat anti-rabbit and donkey anti-mouse IgG HRP-conjugated (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 1% BSA solution. Western Blot was quantified via chemiluminescence using Clarity Western ECL substrate chemiluminescence kit (BioRad). Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software (BioRad).