Ccaat enhancer binding protein alpha c ebpα

Cells were washed twice with phosphate buffered saline (PBS) at 4°C and lysed in RIPA buffer. Protein levels in the cell lysates were measured using a standard Bradford assay. Total protein amount (10 μg) from each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with BSA in Tris-buffered saline, 0.1% Tween^® 20 Detergent (TBST) for 2 hours at RT, the membranes were incubated overnight at 4°C with specific primary antibodies: PPAR-γ (1:1,000; sc-7273; Santa Cruz Biotechnology, Dallas, TX, USA), CCAAT/enhancer binding protein alpha (C/EBPα; 1:500; D56F10, Cell Signaling Technology, Danvers, MA, USA), E-cadherin (1:1,000; ab40772, Abcam, Cambridge, UK), N-cadherin (1:1,000; 13116S, Cell Signaling Technology), Vimentin (1:1,000; 5741S, Cell Signaling Technology), Snail (1:1,000; Novus, NBP2-27293, Littleton, CO, USA), p-P38 (1:1,000; 4511, Novus), total-P38 (1:1,000; 9212, Novus), MMP-2 (1:1,000; GTX-104577, Genetex, Irvine, CA, USA), and β-actin (1:1,000; ab8227, Abcam). The membranes were then washed and incubated with the secondary antibodies goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG (1:2,000; Cell Signaling Technology) for 2 hours with BSA in TBST. The bands were detected using an image analyzer system (Syngene, Cambridge, UK).