Trim66

Primary antibodies were obtained from the following companies: (1) TRIM66, PCNA, caspase7, caspase9, MMP2, MMP19, Twist11, CDH1, Snail1, Snail2, ZEB1 and TGF-β1, Abcam; (2) p-Smad4, Thermo Fisher; (3) p53, p-Smad2, Smad2, Smad4 and GAPDH, CST Biotech. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody was purchased from Beyotime.
Treated and untreated cells were lysed in RIPA lysis buffer with fresh added protease inhibitor cocktail (Sigma). Protein concentration was assayed using BCA protein assay (Thermo Fisher). Equal amounts of protein were then subjected to SDS gel electrophoresis. Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane and immunoblotted with the respective antibodies. Signals were developed by enhanced chemiluminescence (ECL, Millipore). Band intensities were measured using Image J (NIH) and normalized to GAPDH.

Cells of different groups were collected, then lysed using radioimmunoprecipitation buffer containing protease inhibitor. Equal amounts of proteins were loaded and separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (10%) followed by being transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with bovine serum albumin (5%) at room temperature for 2 h, membranes were then incubated with corresponding primary antibodies probing TRIM66 (1:1300; Abcam, Cambridge, MA, USA) and β-actin (1:2400; Santa Cruz, CA, USA) at 4°C overnight. After incubation with corresponding horse radish peroxidase-conjugated secondary antibodies (1:2500; Santa Cruz, Dallas, TX, USA) at room temperature for 2 h, the blots were visualized using an ECL kit (Thermo Fisher, Waltham, MA, USA).