Eif4g1

Cell lysates were prepared by incubation for 5 min on ice with lysis buffer (RIPA buffer with 1% SDS and 1× protease inhibitor cocktail) and sonication for 3×8s pulses in a Soniprep 150 MSE, 30% power. Cell lysates were centrifuged at 4°C, 12000g for 15 min to remove debris. The protein concentration of the supernatants was determined by Pierce BCA Protein Assay Kit (Thermo Scienti c, #23227). Equal amounts of proteins were separated in SDS-PAGE by electrophoresis and transferred to a PVDF membrane (Millipore, #ISEQ00010). The membrane was blocked with 5% skim milk (Solarbio, #D8340) at room temperature and incubated with indicated antibodies overnight at 4°C. And then, the membrane was washed by TBST (1×TBS containing 0.05% Tween-20). The membrane was incubated with a corresponding secondary antibody according to the primary antibody at room temperature for 1h and washed as mentioned above. The blots were visualized using Super ECL Prime (US EVERBRIGHT ® INC, #S6008) by G-box (Syngene, Chemi XT). Intensity of Western blotting band was analyzed by ImageJ 1.46 software, and β-actin was used as a loading control to normalize the amount of protein. Primary antibodies used were antibodies against SNAIL (Cell Signaling Technology, #3879S), E-cadherin (BD Biosciences, #610181), β-actin (Abways Technology, #ab0035), and EIF4G1 (Proteintech, # 15704-1-AP).