E caderin

Primary antibodies for Nodal, fibronectin, E-Caderin, vimentin, Snail, Slug, Zeb1, Twist, YY-1, HIF-1α, Gli1, and GAPDH were purchased from from Abcam (Abcam, Cambridge, UK). Primary antibody for ATM and CSN2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated secondary antibodies were purchased from Bio-rad Laboratories Inc. (Hercules, CA). Cells or tissue samples were re-suspended in ice-cold cell lysis buffer (Cell Signalling Technology) for 20 min to get the protein in supernatant. Each lane in 4-20% Tris-Glycine Gel (Invitrogen) was loaded with 20 μg of protein. After separation, proteins were transferred to PVDF membrane (Millipore, Bedford, MA, USA), blocked with 10% skim milk in Tris-buffered saline with 0.05% Tween-20 for 1 h at room temperature, and incubated with each primary antibody against its specific protein overnight at 4 °C. After washed three times according to standard procedures, membranes were incubated with a 1: 5,000 dilution of the secondary antibodies for 1 h. The bands were detected with ECL chemiluminescence. GAPDH was used as the loading control. Densitometric analysis was performed by use of ImageJ software.