Cells were lysed in lysis buffer and protein concentrations of the lysates were determined using the Bradford protein assay system (Bio‐Rad, Hercules, CA, USA). Equal amounts (30μg protein each lane) of total cellular protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The membrane was blocked and then incubated with primary antibody overnight at 4°C. Primary antibodies used included antibodies for ING5 and SMAD3 (Proteintech Group, Inc., Rosemont, IL, USA); IL‐6 (Bioworld Technology Co., Ltd., Nanjing, China); pAKT (Ser473/Thr308) and STAT3 (Cell Signaling
Technology, Danvers, MA, USA); p‐STAT3 (Y705), AKT, β‐catenin, p‐β‐catenin (Ser33/S37), E‐cadherin, N‐cadherin, Snail, Slug, Twist, EGFR, and CEACAM6 (Abcam, Cambridge, MA, USA); and β‐actin (Actin) (Sigma‐Aldrich, St. Louis, MO, USA). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (1:5000, Sigma‐Aldrich) at room temperature for one hour. The blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham Bioscience, Buckinghamshire, UK).
Technology, Danvers, MA, USA); p‐STAT3 (Y705), AKT, β‐catenin, p‐β‐catenin (Ser33/S37), E‐cadherin, N‐cadherin, Snail, Slug, Twist, EGFR, and CEACAM6 (Abcam, Cambridge, MA, USA); and β‐actin (Actin) (Sigma‐Aldrich, St. Louis, MO, USA). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (1:5000, Sigma‐Aldrich) at room temperature for one hour. The blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham Bioscience, Buckinghamshire, UK).