Ab78016

Total protein from cells was extracted by lysis and resolved by 8–12% SDS-PAGE. Samples were then electro-transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Boston, MA, USA). PVDF membranes were blocked with 5% BSA or skimmed milk. Target proteins were detected with antibodies against GAPDH (Abcam, ab9485, diluted 1:2500), NOX1 (Abcam, ab78016, diluted 1:1000), NOX2 (Abcam, ab31092, diluted 1:1000), NOX3 (Abcam, ab82708, diluted 1:2000), NOX4 (Abcam, ab133303, diluted 1:2000), NOX5 (Abcam, ab198213, diluted 1:2000), DUOX1 (Abcam, ab178534, diluted 1:5000), DUOX2 (Abcam, ab170308, diluted 1:500), VEGFR-1 (Abcam, ab32152, diluted 1:2000), VEGFR-2 (Abcam, ab11939, diluted 1:1000), VEGFR-3 (Abcam, ab27278, diluted 1:1000), EGFR (Abcam, ab52894, diluted 1:5000), p-EGFR (Abcam, ab40815, diluted 1:2000), PDGFR-α (Abcam, ab203491, diluted 1:1000), PDGFR-β (Abcam, ab32570, diluted 1:10000) and C-Met (Abcam, ab51067, diluted 1:5000) overnight at 2–8 °C, followed by incubation with specific horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam, ab6721, diluted 1:10,000) for 2 h at room temperature. Bands were visualized by chemiluminescence with enhanced chemiluminescent detection reagents (Millipore, Boston, MA, USA).

Antibodies composed of Rabbit anti‐human Glutathione Peroxidase 4 (GPX4, Abcam Cat#ab125066, RRID: AB_10973901), Rabbit anti‐human NOX1 (Abcam Cat# ab78016, RRID: AB_1566505), Rabbit anti‐human FACL4 (Abcam Cat# ab155282, RRID: AB_2714020), Rabbit anti‐CD4 (Abcam Cat# ab183685, RRID: AB_2686917), Rabbit anti‐CD8 (Abcam Cat# ab93278, RRID: AB_10563532), and Rabbit anti‐CD86 (Abcam Cat# ab119857, RRID: AB_10902800).

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.