Protein Extraction and Western Blot Analysis

The protein was extracted from the cultured cells and frozen tissue samples using RIPA lysis buffer and protease inhibitor cocktail as described previously [30 (link)]. The same amount of protein was loaded onto the gel using the modified Bradford method for protein quantitative analysis. After being separated by SDS-PAGE, the proteins were electrotransferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010). The membrane was blocked with 5% nonfat milk for 2 h. Finally, the membranes were incubated with the primary antibody overnight at 4 °C. The antibodies used were as follows: SF3B4 (Proteintech, 1:1000, 10482-1-AP), MMP1 (Proteintech, 1:1000, 10371-2-AP), E-cadherin (Proteintech, 1:500, 20874-1-AP), vimentin (Proteintech, 1:1000, 10366-1-AP), Twist1 (Proteintech, 1:1000, 25465-1-AP), ZO-1 (Proteintech, 1:500, 21773-1-AP), KLF16 (Abcam, 1:500, ab187973) and β-actin (Cellsignal, 1:1000, sc-47778). After reaction with HRP-labeled secondary antibody (1:10000, Rockland), the membrane was treated with Immobilon™ Western chemiluminescence HRP substrate (Millipore) and detected by ECL (enhanced chemiluminescence) Fuazon Fx (Vilber Lourmat).