Scl7a11

Cells were lysed in RIPA buffer that contained a protease/phosphatase inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min and used for immunoblotting. The cytosolic fraction for cathepsin B detection was prepared as previously described^{45 (link)}. A total of 20 μg of protein was loaded per lane of an SDS-PAGE gel and separated by electrophoresis. Proteins were transferred onto nitrocellulose (GE Healthcare, IL, USA) or PVDF membranes (IPVH00010, Millipore, MA, USA). Membranes were subsequently probed with primary antibodies and incubated with either goat anti-mouse IgG (Cell Signalling, 7076, MA, USA) or goat anti-rabbit IgG (Cell Signaling,7074) secondary antibodies conjugated with horseradish peroxidase (HRP). Chemiluminescence was detected using an enhanced chemiluminescence (ECL) system (Translab). The following primary antibodies were used: GPX4 (ab125066, Abcam), SCL7A11 (Cell Signalling, 12,691), Nrf2 (ab62352, Abcam), Keap1 (Cell Signalling, 8047), HO-1 (Cell Signalling, 5853), p62 (ab56416, Abcam), LC3 (PM036, MBL), cathepsin B (Cell Signalling, 31,718) and β-actin (Santa Cruz Biotechnology, sc-47778). The result of gels images was cropped and full-length gels and blots are included in the Supplementary Data.

Total protein extracts and Western blot (WB) analysis were performed as in previous studies27 (link). Primary antibodies dilutions were performed as the following: SCL7A11 (#12691, Cell Signaling), Beta-actin (a5441, Sigma-Aldrich). All antibodies were used at 1 mg/mL working concentration in TBST with 5% BSA. The membrane was further probed with horseradish peroxidase (HRP)-conjugated rabbit antimouse immunoglobulin G (IgG) (Santa Cruz Biotechnology, 1:2,000), and the protein bands were visualized using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA).