Tfr antibody

bEnd.3 cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer containing cocktail protease inhibitors (Bio-Rad, Hercules, CA, United States) and boiled with Laemmli buffer and 10% 2-mercaptoethanol (Bio-Rad, Hercules, CA, United States). Blots were probed with anti-transferrin receptor (TfR) antibody (1:1,000 dilution; Thermo Fisher Scientific, Waltham, MA, United States) or anti-ferroportin antibody (1:1,000 dilution; Novus Biologicals, Littleton, CO, United States) overnight at 4°C. Membranes were exposed to the appropriate horseradish peroxidase–conjugated secondary antibodies, followed by chemiluminescence detection (Thermo Fisher Scientific, Waltham, MA, United States). Equal protein loading was controlled by re-probing the membrane with anti–β-actin antibody (1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, United States). Chemiluminescence was detected using the UVP ChemiDoc-It TS2 Imager (Upland, CA, United States), and Image J (NIH, Bethesda, MD, United States) was used for Western blot signal quantification.

Western blot analyses were performed as previously described (44). Antibodies including T-FTH, AKT, p-AKT were from Cell Signaling Technology Inc. (Danvers, MA); DMT1 and FPN antibodies were from Alpha Diagnostic Intl. Inc. (San Antonio, Texas), TfR antibody was from Thermo Fisher Scientific Inc. (Grand Island, NY). DcytB was from Novus Biologicals (Littleton, CO), and GAPDH was from Santa Cruz Biotechnology (Santa Cruz, CA). The western blot results were quantified using ImageJ V1.51h software developed by Wayne Rasband from National Institutes of Health.

The culture medium including DMEM and fetal bovine serum were purchased from Gibco Life Technology Invitrogen (Grand Island, NY, USA). Oligo-fectamine, MEMI, fluo-4 AM, sodium-binding benzofuran isophthalate (SBFI) AM, G-agarose bead, TFR antibody, β-actin antibody, GFAP antibody, DMT1 antibody and DMT1 siRNA duplex chains were from Thermo Fisher Scientific (Waltham, MA USA); siRNA duplex chains of TFR, NCX1-3, Cav-3 and Dab2, NCX2 antibody, Na⁺/K⁺-ATPase alpha1/2 antibody and the secondary antibodies were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NCX1 antibody, NCX3 antibody and native mouse apo-transferrin (apo-TF; i.e., iron-free) were from Abcam (Cambridge, MA, USA). Donkey serum, xestospongin C (Xe-C), nifedipine, ferrous sulfate heptahydrate (FeSO₄), sulforhodamine 101 (SR101) and ferric ammonium citrate were purchase from Sigma-Aldrich (St. Louis, MO, USA). Ryanodine and KB-R7943 were purchased from Calbiochem (La Jolla, CA, USA). Secondary antibody staining with donkey anti-mouse or anti-rabbit Cy-2/3 were from Jackson Immuno-Research (West Grove, PA, USA). Primary antibody of histone H3 was purchased from EarthOx (Millbrae, CA, USA).