Ab172130

BEAS-2B cells were collected and lysed with 0.5 μL RIPA lysis buffer (Beyotime Institute of Biotechnology) on ice for 30 min. Then, proteins were detected using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). 30 µg of protein samples were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C at 1:1000 dilution. On the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG,1:5000, ab172130, Abcam). The signals were detected using enhanced chemiluminescence reagent (GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to analyze the fold-changes of protein levels. The information of primary antibodies were as follows: anti-SLC7A11 (ab175186, Abcam, UK), anti- GPX4 (ab125066, Abcam, UK), anti- FTH1 (ab75972, Abcam, UK), anti- FPN1 (ab78066, Abcam, UK) and anti-GAPDH (ab8245, Abcam, UK).

A549 cells were collected and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) at 4°C for 30 min. Then proteins were detected using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). Loading buffer was added to cytosolic extracts, and after boiling for about 5 min, 30 μg of protein of each sample were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the total gel was transferred into polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 10% skimmed milk for 2 h at room temperature, followed by incubation with anti- SLC7A11 (ab175186, Abcam, UK), anti- GPX4 (ab125066, Abcam, UK), anti- FTH1 (ab75972, Abcam, UK), anti- NOX1 (ab78016, Abcam, UK) and anti-GAPDH (ab8245, Abcam, UK) primary antibodies overnight at 4°C with 1: 1,000 dilution followed by incubation with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG,1:5,000, ab172130, Abcam). The signals were detected using enhanced chemiluminescence reagent (GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to analyze the fold-changes of protein levels.