Anti mouse or anti rabbit hrp conjugated secondary antibodies

Whole cell proteins were extracted with RIPA buffer (Applygen Technologies Inc., China). The protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Pierce Perbio Science). Equal amounts of cell protein were separated using 10% SDS-PAGE gels and the proteins were transferred onto PVDF membranes. Membranes were blotted using a rabbit anti-ABCG1 monoclonal antibody (2,000× dilution; Abcam) followed by anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (10,000× dilution, Santa Cruz, CA, USA) and visualized using an enhanced chemiluminescence technique (ECL, Amersham Biosciences, California, USA). The intensity of the bands was analyzed using the Image J scanning software.

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).