Western Blotting

Cells (LO2, BEL-7402, SMMC-7721, and HepG2) and tissues (HCC and adjacent non-tumor tissues) were lysed in a loading buffer (1×). After lysis, protein extracts (10 μL/lane) were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membrane was blocked in TBST (0.05% Tween 20 in Tris-buffered saline) containing 5% milk for 2 h and then probed with antibodies for NOX4 (Absin Bioscience, Shanghai, China) and FLVCR1 (ABclonal Technology, Wuhan, China), followed by incubation at 4°C for overnight. The membrane was then washed three times with TBST, followed by incubation with secondary antibodies for 1 h at room temperature. Immunoreactive bands thus obtained were visualized by enhanced chemiluminescence.