Ab269514

Proteins were extracted, and the concentration was determined according to standard protocols of protein extraction and BCA protein assay kits, respectively (Sangon Biotech, Shanghai, China). The total protein in the supernatants (30 μg/well) was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-hepcidin (1 : 5000; no. ab190775, Abcam, Cambridge, MA, USA), anti-TfR1 (1 : 5000; no. ab269514, Abcam), anti-Ferritin (1 : 5000; no. ab75973, Abcam), anti-DMT1 (1 : 1000; no. ab157208, Abcam), anti-ALK2 (1 : 500; no. ab262699, Abcam), anti-BMP6 (1 : 1000; no. ab155963, Abcam), anti-SMAD1/5/8 (1 : 1000; orb162781, Biorbyt, UK), and anti-GAPDH (1 : 5000; no. ab8245, Abcam) antibodies. After three washes with TBST, the immunoblots were incubated with alkaline phosphatase-labeled goat anti-rabbit antibodies (1 : 1000, Cell Signaling Technology, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software 1.47 (National Institutes of Health, Bethesda, MD, USA).

After the total protein was extracted, the protein concentration was examined. With 50 μg of total protein, each protein sample was separated for electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. Then, the membranes would be blocked for 2 h at room temperature and incubated with the primary antibody against FPN1 (bs-4906R, Bioss, China), GPX4 (ab125066, Abcam, UK), ACSL4 (ab155282, Abcam, UK), FTH1 (ab183781, Abcam, UK), TFR1 (ab269514, Abcam, UK), xCT (DF12509, Affinity Bioscience, China), 4-HNE (GTX01087, GeneTex, USA), Nox4 (380,874, Zen-bioscience, China), and β-actin (380,624, Zen-bioscience, China) at 4 °C overnight. Then, the membranes were incubated with the second antibodies (Zen-bioscience, China) for 1 h at room temperature. The target bands were visualized by enhanced chemiluminescence and analyzed on Chemiluminescent gel imaging (MiniChemi 610 Plus, Beijing Saizhi Venture Technology Co., Ltd.), which were then quantified by ImageJ analysis software (National Institutes of Health, Bethesda, MD).