Ab1086

Three iron transport-related proteins (DMT-1, TfR-1, and FP1) were detected by Western blotting. Frozen heart tissue samples were homogenized with 400 μL lysis buffer/20 mg tissue and then centrifuged at 12,000 r/min for 10 min, after which the supernatant was collected. Total proteins were loaded and separated on a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and then transferred to a nitrocellulose membrane. Blocking was performed with 5% skimmed milk powder in phosphate buffer saline (PBS), and then the membranes were incubated with primary antibodies overnight at 4°C. The membrane was washed three times (10 min each) to remove uncombined primary antibodies, and then it was incubated with secondary antibodies at room temperature for 90 min. After the membrane was washed three times to remove unbound secondary antibodies, the membrane was exposed and scanned by the Gel imaging analysis system (UVP, USA), and the gray value was automatically measured by the system. The primary antibodies included anti-DMT1 antibody (ab55735, Abcam), anti-transferrin receptor antibody [MEM-189] (ab1086, Abcam), and anti-ferroportin/SLC40A1 antibody (ab58695, Abcam).

The tumor and A549 cell lysates were prepared, washed with cold PBS, resuspended in a lysis buffer and sonicated the lysate. The proteins were separated on 10–15% SDS gels and transferred to nitrocellulose membranes. After incubation with 1:1,000 primary antibody dilution buffer for 1 h, Goat Anti-Mouse IgG (H + L) HRP (1:5,000, No. S0002, Affinity) or Goat Anti-Rabbit IgG (H + L) HRP (1:5,000, No. S0001, Affinity) were used as secondary antibodies and developed by enhanced chemiluminescence. The antibodies used for immunoblotting were ubiquitin-specific peptidase 14 (USP14, 1191S, CST, USA), Beclin 1 (3,738, CST, USA), nuclear receptor coactivator 4 (NCOA4, ab86707, Abcam, United Kingdom), microtubule-associated protein light chain 3 (LC3 I and LC3 II 12741, CST, USA), ferritin heavy chain 1 (FTH1, 3,998, CST, USA), transferrin receptor 1 (TfR1, ab1086, Abcam, United Kingdom), glutathione peroxidase 4 (GPX4, ab125066, Abcam, United Kingdom), activating transcription factor4 (ATF4, ab184909, Abcam, United Kingdom) and β-tubulin (2146S, CST, USA).