| Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacture's protocol. Briefly, the cells were incubated with the lysis buffer at room temperature for 5 min. Then cell lysates were centrifuged at 13,000 × g for 5 min at room temperature and supernatants were harvested. |
Equal amounts of total protein (20 µg) were separated using 12% SDS-PAGE and subsequently transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at 4°C overnight, membranes were incubated with keratin rabbit polyclonal antibody (1:400; cat. no. 41723; Signalway Antibody Inc., College Park, MD, USA), GSTA1 monoclonal antibody (1:500; cat. no. sc-100546), E-cadherin rabbit polyclonal antibody (1:500; cat. no. sc-7870), vimentin monoclonal antibody (1:800; cat. no. sc-373717), N-cadherin mouse monoclonal antibody (1:500; cat. no. sc-393933) and β-actin monoclonal antibody (1:1,000; cat. no. sc-130301; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase-labeled goat anti-mouse (1:2,000; cat. no. sc-2005) or goat anti-rabbit antibodies (1:2,000; cat. no. sc-2004; both Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. |
Immunoreactive bands were detected using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.). ImageJ version 1.37 software (National Institutes of Health, Bethesda, MD, USA) was used for quantification, and the experiments were independently performed three times. |