Western Blot Analysis of Protein Expression

Cells were lysed on ice, and the total protein was collected. The bicinchoninic acid (BCA) method was used to determine the protein concentrations. Western blot assay was performed as previously described [14 (link), 15 (link)]. Proteins were transferred to polyvinylidene fluoride membranes (Merck Millipore, MA, USA) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were closed with 5% bovine serum albumin (BSA) in TBST for 1 h at room temperature and then incubated with primary antibody overnight at 4°C. Subsequently, the membranes were incubated with the second antibodies for 1 h at room temperature. The immunoreactive bands were detected via chemiluminescence (ECL Plus, Merck Millipore). Antibodies were as follows: c-MET Rabbit mAb (ABclonal, #A17366, 1 : 1000), SLC7A11/xCT Rabbit mAb (ABclonal, #A2413, 1 : 1000), GPX4 Monoclonal antibody (Proteintech, #14432-1-AP, 1 : 1000), and Anti-beta Tubulin antibody (Abcam, #ab6046, 1 : 1000). The secondary antibodies (either antirabbit or antimouse) were obtained from Thermo Fisher Scientific (Invitrogen).