Western blotting and Cycloheximide (CHX) Chase Assays

Total proteins were extracted from HCC tissue samples or cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors. The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology), and proteins were separated on polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Then, membranes were blocked with 5% skimmed milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. After washing with TBST three times, the membranes were incubated with secondary antibodies conjugated to HRP (Cell Signaling Technology) for 2 h at room temperature. Finally, proteins were visualized using an ECL Plus kit (Bio-Rad, Hercules, CA, USA). The relative protein expression was analyzed using Quantity One software (Bio-Rad).

To examine the half-life of TGFR-1, Cycloheximide (CHX) Chase assay was performed. SK-Hep1-shRNA/USP4 cells or SK-Hep1-USP4 cells were treated with protein synthesis inhibitor CHX (100 µg/mL) for the indicated times. Then, proteins were extracted and Western blotting was performed.