Ab55735

As previously described (Zhang M. et al., 2020 (link)), tissue slices were obtained at 24 h, 3 days, and 21 days, and treated with IRP1/DMT1/4’,6-diamidino-2-phenylindole (DAPI) and micro-tubule-associated protein light chain 3B (LC3B)/translocase of outer mitochondrial membrane 20 (TOMM20)/DAPI immunohistochemical staining. Rabbit monoclonal antibodies against IRP1 (1:200, ab236773, Abcam, UK) and LC3B (1:200, ab48394, Abcam, UK) and mouse monoclonal antibodies against TOMM20 (1:200, ab56783, Abcam, UK) and DMT1 (1:200, ab55735, Abcam, UK) were used for immunohistochemistry. Images were obtained using a fluorescence microscope (Olympus, Japan) and analyzed using ImageJ V.1.37 software (National Institutes of Health, USA).

Cells or tissue samples were lysed in RIPA lysis buffer (Tian P. et al., 2017 (link)) added protease inhibitor (P8340, Sigma, St. Louis, MO, United States) for 30 min on ice, and then centrifuged at 12, 000 rpm for 15 min at 4°C. The supernatant was collected and measured to calculate protein concentration using a BCA protein assay kit (23225, Thermo Fisher Scientific, Waltham, PA, United States). After denaturation, 80 μg protein was electrophoresed in a 10% SDS-PAGE, and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk powder in TBST (Tris buffer with 0.1% Tween, pH 7.6) for 2 h and then incubated at 4°C overnight with primary antibodies: DMT1 (ab55735, Abcam, 1:500) or β-actin (BS6007M, Bioworld, 1:10000). A secondary HRP-conjugated antibody (BS12478, Bioworld, 1:10000) was used to incubate the membrane for 2 h prior to chemiluminescent detection. The signals were determined using a chemiluminescent substrate (ECL) kit (NCI4106, Thermo Fisher Scientific, Waltham, PA, United States). Then, protein intensities were quantified by the VersaDoc MP 4000 system (Bio-Rad, California, CA, United States).

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).

Freshly isolated mouse retinas were homogenized and lysed in ice-cold Radioimmunoprecipitation assay (RIPA) buffer (P0013; Beyotime). A Bicinchoninic Acid Kit (Pierce, Rockford, IL, USA) was used to quantify the total protein content. Western blotting was performed as described in our previous study [35 (link)] using the following antibodies: Anti-ACSL4 (as used in IF; 1:3000), anti-GPX4 (as used in IF; 1:1000), anti-FTH1 (as used in IF; 1:1000), anti-divalent metal transporter 1 (DMT1; ab55735; Abcam; 1:1000), anti-transferrin receptor (TR; ab84036; Abcam; 1:1000), anti-ferroportin 1 (Fpn1; ab235166; Abcam; 1:500), anti-nuclear receptor coactivator 4 (NCOA4; sc-373739; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:500), and anti-β-actin (GB12001; Servicebio; 1:3000). The original western blots for these results are provided in Supplementary File 1.

Mouse colonic tissues and ECs were lysed using the radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a mixture of protease inhibitors to obtain protein samples, and samples were centrifugated and the protein concentration was examined by a bicinchoninic acid kit (P0011, Beyotime). Equal amounts of proteins were received sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes, and after blockade with defatted milk, the membranes were incubated with primary antibodies against GPX4 (1:1000, ab125066, Abcam), IRF7 (1:1000, ab288440, Abcam), SLC11A2 (1:1000, ab55735, Abcam), and β-actin (1:5000, ab6276, Abcam) and with appropriate amount of horse radish peroxidase-conjugated secondary antibody IgG (1:1000, ab6721, Abcam). Protein signals were visualized using the enhanced-chemiluminescence reagent (P0018, Beyotime).

Cryoprotected retinal sections (10-μm thick) were prepared and fixed in 4% paraformaldehyde (PFA), followed by incubation with rabbit antibodies against the transferrin receptor (Tfrc, ab214039; Abcam), ferritin light chain (Ftl, ab69090; Abcam), ferritin heavy chain (Fth, ab65080; Abcam), divalent metal transporter (Dmt1, ab55735; Abcam), and ferroportin (Fpn-1, NBP1-21502, Novus Biologicals, Littleton, CO, USA) and secondary antibodies labeled with Alexa Fluor 488 (Thermo Fisher Scientific) and DAPI (Invitrogen). The sections were analyzed using a fluorescence microscope (BZ-9000; Keyence Corporation of America). To detect and visualize ⁵⁷Fe localization and its relation with cone photoreceptor cells in the retina via LA–ICP–MS, retinal sections of ⁵⁷Fe IVT mice were fixed with 4% PFA and incubated with an anti-opsin antibody (AB5405, 1:200, Chemi-Con, Temecula, CA), followed by incubation with a colloidal gold-conjugated goat anti-rabbit polyclonal secondary antibody (1:20, Jackson ImmunoResearch Laboratories, West Grove, PA).