Western Blot Analysis of Signaling Proteins

The cells were lysed in RIPA buffer as described previously [4 (link)]. Approximately 20–80 μg aliquots of protein were subjected to SDS–polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were incubated with anti-p-AKT (9271, 1:1000, Cell Signaling, Danvers, MA, USA), anti-AKT (sc-8312, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-catenin (sc-7199, 1:2000, Santa Cruz Biotechnology), anti-Snail (3895, 1:1000, Cell Signaling), anti-vimentin (sc-6260, 1:1000, Santa Cruz), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich) antibodies, and the bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) Western blotting detection reagent (170-5061, Bio-Rad Laboratories, Hercules, CA, USA).