Rabbit anti human gapdh primary antibody

Total proteins were extracted from the five cell lines (GES-1, NCI-N87, SGC7901, BGC823 and HGC-27), and protein concentration was determined by Bradford assay. Protein samples were then separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene fluoride membrane using a Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% skimmed milk powder at room temperature for 90 min, then incubated with rabbit anti-human KLF17 polyclonal primary antibody and rabbit anti-human GAPDH primary antibody as the loading control (1: 400 dilution; Abcam, Shanghai, China) overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Abcam) at 37°C for 1 h. After rinsing, the immunosignal was developed in a dark room and the membrane image was captured using a Labworks 4.6 system (Labworks, Novato, USA). The signal intensity (grey value) was then read with Image J software, and average absorbance of the protein bands was used to indicate the intensity of protein expression.

Total protein was extracted from cultured cells or tissue samples using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, People’s Republic of China) in the presence of a protease inhibitor cocktail (Pierce Biotechnology, Inc., Rockford, IL, USA). A bicinchoninic acid protein assay (Aidlab Biotechnologies Co., Ltd., Beijing, People’s Republic of China) was conducted to determine the concentration of total protein. Equal amounts of protein were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes (Sigma-Aldrich Co., St Louis MO, USA), and then blocked at room temperature for 1 hour in 5% fat-free milk diluted in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-human ZEB1 primary antibody (1:1,000 dilution; cat no: ab203829) or rabbit anti-human GAPDH primary antibody (1:1,000 dilution; cat no: ab181603; both from Abcam, Cambridge, MA, USA). After washing three times with TBST, horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000 dilution; cat no: ab205718; Abcam) were incubated with the membranes at room temperature for 2 hours. Finally, an ECL Protein Detection kit (Pierce Biotechnology, Inc.) was applied to develop the protein bands.