Anti human phospho p70s6k

The cells were harvested by centrifugation and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The anti-human E-cadherin, anti-human vimentin, anti-human SNAILl2, anti-human STAT3, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-human β-actin primary antibodies and Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China). After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences, New York, NY, USA).