Radiolabeled 59fecl3

HepG2 cells (ATCC) were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Plasmid encoding DMT1-GFP (-IRE) was from Jerry Kaplan (University of Utah). PrP^C and PrP^C-GFP constructs were prepared by ligating cDNA from previously reported constructs [24 (link)] in modified PiggyBac vectors (System Biosciences, Mountainview, CA). HepG2 cells were co-transfected with the PiggyBac vectors and a transposase-expression vector using Lipofectamine® LTX (Invitrogen, Carlsbad, CA), and mixed cultures of ~30 stable clones selected with Blasticidin-S were used for all experiments to avoid clonal artifacts. PrP antibody specific for residues 109-112 (3F4) was from Signet Laboratories (Dedham, MA). The specificity of anti-C antibody against C-terminal residues of human PrP (anti-C) has been described previously [25 (link)]. Antibodies specific to ferritin and β-actin were obtained from Sigma (Sigma-Aldrich, St. Louis, MO) and Millipore (Bedford, MA) respectively. Wheat Germ Agglutinin (WGA)-Alexa Fluor-594 was from Molecular Probes (Invitrogen). HRP-conjugated secondary antibodies and ECL Detection Kit were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Radiolabeled ⁵⁹FeCl₃ was from Perkin-Elmer (Boston, MA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).