Tetra handcast system

According to the manufacturer’s instructions, proteins were extracted from cells or tissues using Western-IP Lysis Buffer (Beyotime, Shanghai, China). The protein concentration was determined using a bicinchoninic acid (BCA) protein concentration determination assay kit (Beyotime, Shanghai, China). Prepared samples were electrophoresed in a 10% SDS-PAGE gel and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the Tetra Handcast system (Bio-Rad, USA). The membranes were blocked for 3 h at room temperature and incubated overnight at 4°C with an appropriate primary antibody in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. After washing in TBST, the membranes were incubated with secondary antibody, washed and visualized using a supersensitive enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (SYNGENE, UK).
Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).

Total proteins were extracted from fresh tissues and cells using RIPA Protein Lysis solution (Pierce, IL, USA) and quantified by the Bradford method. Prepared samples were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a polyvinylidene fluoride membrane (Millipore, MA, USA) using the Tetra Handcast system (Bio-Rad, USA). The membrane was blocked for 1 h at room temperature or overnight at 4 °C in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. Then it was incubated overnight at 4 °C with the appropriate primary antibody, and followed by the secondary antibody for 70 min at room temperature. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (Syngene, UK) with an electrochemiluminescence kit (Pierce, IL, USA).
The primary anti-SOX3 antibody was purchased from Abcam (MA, USA), and the anti-β actin antibody was purchased from Santa Cruz Biotechnology Inc. (CA, USA). Other primary antibodies (anti-E-cadherin, anti-CK-18, anti-N-cadherin, anti-vimentin, anti-Snail1, anti-Twist1, anti-Slug, anti-ZEB1, anti- ZEB2, anti-FLAG) were purchased from Cell Signaling Technology (MA, USA). Anti-mouse or anti-rabbit HRP-conjugated secondary antibodies were purchase from Santa Cruz Biotechnology Inc. (CA, USA).