Anti zeb2

The proteins were extracted from the whole cell lysates using RIPA cell lysis buffer and the protein concentration was determined by the Bradford reagent. In total, 20 μg of the extracted total cellular protein from each sample were separated via SDS-PAGE, and transblotted onto EMD Millipore Immobilon™-P PVDF Transfer Membranes (EMD Millipore Cat No.: IPVH00010, USA). Western blot analyses were conducted with the following antibodies: mouse monoclonal anti-CD109 (Cat. No. 556039, BD Biosciences), anti-fibronectin (Cat. No. 610078, BD Biosciences), anti-Vimentin (D21H3) XP^® Rabbit (Cat. No. 5741. Cell signaling); Anti-ZEB2 (Cat. No. sc-271984, Santa Cruz Biotechnology); rabbit polyclonal anti-Snail (Cat. No. ab5351, Abcam), anti-twist (Cat. No. ab505181; Abcam), anti-TGFBR1 (ALK5) (Cat. No. AHO1552, Invitrogen), anti-N Cadherin (Cat. No. ab18203, Abcam), anti-E-Cadherin (Cat. No. ab216783, Abcam), Anti-P-Smad2 (Cat. No. mAb #3108, cell signalling); Anti-P-Smad2/3 (Cat. No. MAB8935, R&D system, and anti- Slug (Cat. No. 9585S Cell signaling), anti-β-actin antibodies (Cat. No. sc-47778, Santa Cruz Biotechnology).

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).