Mike Rudnicki (University of Ottawa) and ubiquitous Hjv−/− mice (in mixed 129S6/SvEvTac;C57BL/6 background) from Dr. Nancy Andrews (Duke University). Only male animals were used for experiments, housed in macrolone cages (up to 5 mice/cage, 12:12-hour CH5424802 in vitro light-dark cycle: 7 AM to 7 PM; 22 ± 1°C, 60 ± 5% humidity) according to standard institutional guidelines. The mice had free access to water and food, a standard diet containing approximately 226 mg of iron per kg (Teklad Global 18% protein rodent diet, TD 2018, Harlan Laboratories, Indianapolis, IN). Experimental procedures
were approved by the Animal Care Committee of McGill University (protocol 4966). Transferrin saturation, total iron binding capacity (TIBC), serum iron, and ferritin were measured with a Roche Hitachi 917 Chemistry Analyzer at the Biochemistry Department of the Jewish General Hospital. Hepatic and splenic nonheme iron was measured by the ferrozine assay, as described.33, 34 Results are expressed as micrograms of iron per gram of dry tissue weight. Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. To visualize ferric iron deposits, deparaffinized tissue sections were stained with Perls’ Prussian blue using the Accustain Iron Stain kit (Sigma). Total RNA was isolated from frozen tissues using
the RNeasy Mini kit (Qiagen) for the liver and the RNeasy Fibrous Tissue Mini kit (Qiagen) for the skeletal muscles and heart; its quality was assessed by determining the 260/280 nm absorbance ratios and by agarose Selleck R428 gel electrophoresis. qPCR was performed by using gene-specific primers (Table 1B),
as described,35 with β-actin or ribosomal protein S18 (rS18) as housekeeping genes for normalization. Duodenal membrane-enriched protein lysates were prepared as in36 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel; the samples (30 μg of protein) did not contain β-mercaptoethanol Bumetanide and were not boiled before loading on the gel. Following transfer of the proteins onto nitrocellulose filters (BioRad), the blots were saturated with 10% nonfat milk in phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween-20 (PBS-T) and probed with a 1:1,000 diluted ferroportin antibody.35 After three washes with PBS-T, the blots were incubated with 1:5,000 diluted peroxidase-coupled goat antirabbit IgG (Sigma). The peroxidase signal was detected by enhanced chemiluminescence with the Western Lightning ECL kit (Perkin Elmer). Quantitative data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the two-tailed Student’s t test with GraphPad Prism software (v. 5.0d). A probability value P < 0.05 was considered statistically significant. We introduced loxP sites flanking exons 2-4 of the Hfe2 gene (encompassing the entire open reading frame of Hjv messenger RNA [mRNA]) and generated a mouse line carrying floxed Hfe2f/f alleles (Fig. 1A).