Goat anti rabbit or anti mouse igg conjugated to horseradish peroxidase secondary antibody

SGC7901 cells were washed twice in cold PBS and then lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology Jiangsu, P.R. China) containing protease inhibitor cocktail (Millipore). The protein concentration of cell lysates was quantified by BCA kit (Beyotime Institute of Biotechnology), and 50 ng of each of the proteins was separated by SDS-PAGE on 10% gels and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% nonfat milk diluted with TBST at room temperature for 1 h and incubated overnight at 4°C with primary antibodies such as anti-annexin A4, anti-MMP-2, anti-MMP-9, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Twist1, anti-Slug and anti-ZEB1 (1:500; Abcam, Cambridge, UK) and anti-TIMP-1 (1:1,000; Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with a goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase secondary antibody (1:1,000; Cell Signaling Technology) for 2 h. The proteins were visualized using ECL-plus reagents (Amersham Biosciences Corp., Piscataway, NJ, USA). The density of the bands was measured using the ImageJ software (NIH, Bethesda, MD, USA), and values were normalized to the densitometric values of β-actin (1:5,000; Sigma-Aldrich, St. Louis, MO, USA) in each sample.