Phospho fak

The tissue protein was isolated from human liver tisseus using T-PER Tissue Protein Extraction Reagent (Pierce Biotechnology) according to protocols provided by the manufacturer. The cell protein was lysed using lysis buffer that contained Tris-HCl, NaCl, Triton-X 100, MgCl2, PMSF and so on. The cell lysate, which was used for cell signaling detection, was obtained by IP cell lysis solution (Beyotime Biotechnology). All the proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane under constant current condition. Then, the membrane was blocked using 5% bovine serum albumin at room temperature for 1 h. The nitrocellulose membrane was then incubated using antibodies for Smoc2 (Abcam), extracellular regulated protein kinase (ERK; Cell Signaling Technology, CST), phospho-ERK (CST), AKT (CST), phospho-AKT (CST), Src (CST), phospho-Src (CST), FAK (CST), phospho-FAK (CST) and GAPDH (Sigma) at 4 °C overnight. The next day, the nitrocellulose membrane was incubated with the fluorescence-conjugated secondary antibodies at room temperature for 1 h. All the fluorescence signals were captured and saved by the Odyssey imaging system (LI-COR). The images of western blots were analyzed using ImageJ software for gray value calculation.