Ab84036

The western blot assay was carried out as previously described (Zhao et al., 2021 (link)). The primary antibodies against caspase-3 (Abcam, ab65080), cyclin D1 (Abcam, ab226977), CDK4 (Abcam, ab137675), p21 (Abcam, ab227443), FPN (Abcam, ab235166), TF (Abcam, ab84036), FTH1 (Abcam, ab65080), GPX4 (Abcam, ab125066), Nrf2 (Abcam, ab137550), HO-1 (Abcam, ab13243) and β-actin (Abcam, ab8227) were used in this investigation.

Coronal slices were dissected out after the indicated reperfusion time and the right hemisphere (the infarct/ischemic side) was selected. For western blotting, proteins were extracted from mouse brains using lysis buffer (50 mM Tris–HCl, pH 7.4, 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na₃VO₄, 40 mM Na₂P₂O₇·10H₂O, 50 mM NaF, 100 nM calyculin A, 50 µg/mL leupeptin, 25 µg/mL pepstatin A, 50 µg/mL trypsin inhibitor, and 1 mM dithiothreitol) as previously described^{72 (link)}. The protein concentrations were quantified using Bradford’s assay and normalized. Equal quantities of protein were loaded and separated by 4–12% SDS–polyacrylamide gels (SDS/PAGE) and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were probed with primary antibodies overnight at 4°C and then with appropriate secondary antibodies. Thereafter, membranes were detected with an enhanced chemiluminescence (ECL) immunoblotting detection system (Amersham Biosciences, NJ, USA) using Luminescent Image Analyzer (LAS-4000 mini, Fuji Film, Tokyo, Japan). The densities of the bands were analyzed with ImageJ software (NIH, Bethesda, MA, USA). Primary antibodies were: SAA (R&D systems, AF2948); transferrin (Abcam, ab84036); NLRP3 (Abcam, ab214185); actin (Abcam, ab179467). Secondary antibodies were: Goat anti-rabbit (Sigma, A6154).

The following antibodies were used: β-actin antibody (cw0096m, CWbio, China), FPN1 antibody (MTP11-S, ADI, USA), DMT1 (+IRE) antibody (NRAMP21-S, ADI, USA), 4-HNE antibody (HNE-11S, ADI, USA), TfR1 antibody (ab84036, Abcam, USA), L-ferritin antibody (ab109373, Abcam, USA), H-ferritin antibody (ab183781, Abcam, USA), hepcidin antibody (ab30760, Abcam, USA), Aβ_22–35 antibody (A3356, Sigma, USA), phospho-p38 (p-p38) antibody (4511S, CST, USA), p38 antibody (8690S, CST, USA), phospho-ERK (p-ERK) antibody (9102S, CST, USA), ERK antibody (4372S, CST, USA), Bcl-2 antibody (12789-1-AP, Proteintech, China), Bax antibody (50599-2-Ig, Proteintech, China), GFAP antibody (MAB360, Millipore, USA), Iba1 antibody (MABN92, Millipore, USA), NeuN (ab104224, Abcam, USA), CD31 (77699T, CST, USA), PSD-95 (ab18258, Abcam, USA), anti-rabbit IgG (RPN4301, Amersham, UK), anti-mouse IgG (RPN4201, Amersham, UK), DyLight 488 goat anti-mouse IgG (A23210, Abbkine, USA), DyLight 549 goat anti-rabbit IgG (A23320, Abbkine, USA), and DyLight 549 goat anti-mouse IgG (A23310, Abbkine, USA).

Briefly, hCMEC/D3 cells were seeded (45,000 cells/ml) one day before the experiment. The following day, cells were overnight incubated with Cy5-labeled BTL (empty) to a final concentration of 2.5 mM total lipids, corresponding to 2.20×10¹³ liposomes/ml. Subsequently, the culture medium was removed, and cells were thrice rinsed with PBS and fixed with cold 4% paraformaldehyde (PFA) for 10 min. The cells were then permeabilized (0.1% Triton X100 for 5 min), blocked (10% FBS in 0.05% Tween20 in PBS for 30 min), and stained with Anti-Transferrin Receptor Antibody (ab84036; Abcam) at 5 μg/ml in blocking serum for 1 h. Next, cells were stained with Goat Anti-Rabbit IgG H&L conjugated Alexa Fluor 488 (ab150077; Abcam) at a dilution of 1:1000 in blocking serum for 1 h and then thrice rinsed with PBS (Figure S9). Acquisition and processing were performed using super-resolution (SR) microscopy (Elyra 7 eLS, Zeiss, Germany) and ZEN software, applying 405-, 488-, 561-, and 642-nm lasers.

Western blot analysis was performed as previously described (Inoue et al., 2014 (link)). Protein extracts (3 μg) were subjected to SDS-PAGE and separated proteins were transferred onto polyvinylidene difluoride membranes. After blocking with a solution containing 5% skim milk in PBS, the membranes were incubated with rabbit anti-GluA1 (1:2000, catalog #GluR1C-Rb-Af692, Frontier Institute, Hokkaido, Japan) or anti-GluA2 (1:2000, catalog #GluR2C-Rb-Af1050, Frontier Institute) polyclonal antibodies overnight at 4°C, then with an appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected using an ECL chemiluminescence detection system and an Image Quant LAS 4000 Mini (GE Healthcare, Uppsala, Sweden). For reprobing, the membranes were incubated with stripping buffer (62.5 mM Tris-HCl, 2% SDS, and 100 mM 2-mercaptoethanol) for 30 min at 50°C. The membranes were incubated with rabbit anti-transferrin receptor antibody (1:1000, catalog #ab84036, Abcam, Bristol, UK) and then processed as described above. The signal of the protein bands was quantified using ImageJ 1.46r software.

PrP^C-specific antibodies 3F4 and 8H4 were obtained from Signet laboratories (Dedham, MA, USA). 3F4 binds to an epitope on amino acids 109–112 (Human PrP sequence), while 8H4 binds an epitope in the C-terminal amino acids 145–180 of human and mouse PrP^{62 (link)}. Other primary antibodies were obtained from the following sources: anti-ferritin (F5012, Sigma Aldrich, USA), anti-DMT-1 (ab55812, Abcam, USA), anti-TfR (ab84036, Abcam, USA), anti-Tf (GTX21223, GeneTex, USA), and anti-β-actin (MAB1501, Millipore, USA) Secondary antibodies conjugated with HRP were from GE Healthcare (anti-mouse, LNA931V, anti-rabbit, LNA934V). Alexa fluor-conjugated secondary antibodies were from molecular probes, ImmPACT DAB (SK4105) from Vector labs, USA, PNGase F (P0704S) from NEB, USA and Lipofectamine 3000 transfection reagent from Invitrogen, USA. Radiolabeled ⁵⁹FeCl₃ was obtained from Perkin-Elmer, USA. siRNA against PrP (sc36318) and scrambled siRNA (sc37007) were purchased from Santa Cruz Biotechnology Inc, USA. For immunohistochemistry of hamster retinal sections PrP-6C2 (CVI-WUR, Lelystad, Netherland), ferritin (Jackson ImmunoResearch, West Grove, PA, USA), and Iba1 (019–19741, Wako Chemicals, USA) antibodies were used. FAC (F5879) and all other chemicals were from Sigma Aldrich, USA.

From these decalcified samples, 5 μm sagittal sections were cut from the medial knee compartment and were deparaffinized, antigen retrieved, incubated with corresponding primary antibodies TFR1 (ab84036, Abcam, dilution 1:3000), DMT1 (ab55735, Abcam, dilution 1:1000), FPN(ab78066, Abcam, dilution 1:100), MMP13 (ab39012, Abcam, dilution 1:1000), ADAMTS5 (ab182795, Abcam, dilution 1:1000), and then incubated with biotinylated goat anti-rabbit (BA1003, Boster, China, dilution 1:100) secondary antibodies. Sections were colored with DAB and counterstained with hematoxylin. The images of immunohistochemical staining were analyzed using the software Image-Pro Plus. To quantify immuno-positive cells, at least five random fields in the cartilage were selected and the ratio of immune positive cells to total cells were calculated.