Gapdh mab hrp

The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with RIPA 10 µl/ml phenylmethanesulfonyl fluoride (PMSF), a protease inhibitor, and protein concentrations were determined by a Bicinchoninic Acid (BCA) Protein assay kit. All of these reagents were purchased from the Beyotime Institute of Biotechnology, China. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 2 h before being incubated with the appropriate primary antibodies overnight at 4°C. The membranes were subsequently incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. The protein bands were visualized by an Enhanced Chemiluminescence Detection kit (Thermo Scientific, Waltham, MA, USA) and photographed by a Gel Logic 2200 PRO imaging system. The following primary antibodies were used in this experiment: an MTDH rabbit mAb (1:2000, Abcam), an E-cadherin rabbit mAb (1:1000, Cell Signaling Technology, Beverly, MA, USA), a Slug rabbit mAb (1:1000, Cell Signaling Technology), a Snail rabbit pAb (1:1000, Abcam), an N-cadherin mouse mAb (1:1000, Cell Signaling Technology), a ZO-1 mouse mAb (1:2000, Cell Signaling Technology), and a GAPDH mAb-HRP (1:5000, Bioworld Technology, Minneapolis, MN, USA).