Aldh1

Total protein was isolated from human tissue samples and colorectal tumor cells using lysis buffer containing protease inhibitors (Pierce Biotechnology, Waltham, USA). Equal amounts of protein samples were separated on a 10% SDS-PAGE gel and then blotted with the following primary antibodies (Abcam unless indicated otherwise): FBXW11 (1 : 500; catalog number 137835), ALDH1 (1 : 500; catalog number 52492), Nanog (1 : 1000; catalog number 109250), Oct4 (1 : 1000; catalog number 181557), HIC1 (1 : 500; catalog number 271499, Santa Cruz Biotechnology), SIRT1 (1 µg/mL; catalog number 110304), and β-actin (internal control; 1 : 2000, catalog number 32572). After 1 h incubation with a secondary antibody (1 : 2000; catalog number 6721, Abcam), the blots were visualized by the Alphalmager^TM 2000 Imaging System (Alpha Innotech, San Leandro, USA). The band density was quantified using ImageJ.

Whole cell lysates (50 μg) were separated by electrophoresis on 12.5% denaturing polyacrylamide gels. The membranes were incubated overnight at 4°C with primary antibodies (0.1 μg/ml) against Oct4, Nanog, β-AR and phospho EGFR were purchased from Abcam Corporation (Abcam, Cambridge, UK), Snail, Twist, Fibronectin, E-cadherin, CD21, CD44, CD133, ALDH-1, phospho AKT, phospho NFκB, α7-nAChR, Bcl-2 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Bax was obtained from Becton, Dickinson and Company (BD, Transduction Laboratories TM), MDR-1 and ABCG2 were purchased from Millipore Corporation (Millipore Corporation, Billerica, MA, USA), caspase-3 and PARP were obtained from Invitrogen Corporation (Invitrogen, Camarillo, CA, USA) in Tris-Tween-Buffer-Saline buffer containing 3% non-fat milk. Subsequently, each membrane was washed and incubated for 1 h at 25°C with a secondary anti-mouse, anti-rabbit, or anti-goat antibody conjugated with horseradish peroxidase (1: 1000; Santa Cruz Biotechnology, Inc.), as previously reported. [25 (link)]