Ab26096

Protein content in lysates was measured using the Bradford assay. Cell lysates were boiled with loading buffer containing β-Mercaptoethanol at 95 °C for 5–10 min. Approximately 50 µg of protein were loaded onto 10% SDS-polyacrylamide gels and transferred onto PVDF membranes for Western blotting. Membranes were blocked in 5% non-fat dry milk in TRIS–buffered saline containing 0.1% Tween (TBST) for at least 1 h at room temperature, then incubated at 4 °C overnight in primary antibody: NRF2 (Santa Cruz sc-722, 1:1000), KEAP1 (Cell Signaling 546C, 1:1000), ETS-1 (Abcam ab26096">ab26096, 1:1000), or xCT (Abcam ab37185">ab37185, 1:1000). Membranes were washed 3×10 min with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody targeted to rabbit (Cell Signaling 7074S, 1:5000) for 1 h at room temperature. Following washes with TBST, blots were developed using an enhanced chemiluminescence kit (ECL plus, Amersham Biosciences). Either staining with amido black (Sigma) or reprobing stripped blots with calnexin (Santa Cruz H-70, 1:1000) or actin (Cell Signaling 13E5, 1:1000) were used as loading controls. Western Blots were densitometrically quantified using ImageJ software (NIH). Normalized protein levels were then compared by fold change.