Anti acy 1

Lysates from CRC cells were separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and the proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with TBST (0.5% Tween-20 in Tris-buffered saline) containing 5% nonfat milk for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C, followed by secondary antibodies for 1 h at room temperature. The following antibodies were used: anti-GAPDH, anti-ACY1, anti-E-cadherin, and β-catenin (Cell Signaling Technology, Boston, MA, USA). The samples were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Cambridge, MA, USA), and the bands were analyzed with ImageJ software (version 1.43 National Institutes of Health, Bethesda, MD, USA).