Expression patterns of IRP1 and IRP2 determined by in situ hybridization and Western blotting. In situ hybridization was performed using DIG-labeled cRNA probes and AP detection. Purple/brown staining develops in RNA-positive areas. (A) Fresh frozen sections of newborn pups at postpartum day 0 (P0); inset: thymus. Note that IRP1 is highly expressed in the brown fat and liver, whereas IRP2 expression is high in the brain, thymus and retina. (B) Adult brain coronal sections reveal that IRP2 is highly expressed in several brain regions. IRP1 is also expressed in these regions. (C) In adult peripheral tissues, IRP1 is highly expressed in the kidney, liver and epididymis, whereas IRP2 is most highly expressed in the testis. (D) Protein analysis by Western blotting. Lysates from each of the tissues indicated (40 μg protein/lane) were separated by SDS–PAGE, transferred to membranes, and IRP levels were compared between tissues in one gel that is representative of three separate experiments. Note that IRP1 levels are highest in the kidney, liver and brown fat, whereas IRP2 levels are highest in the cerebellum and forebrain and are hardly detectable in the brown fat.

IRP1 activity levels in tissues determined by aconitase and gel retardation assays. (A) Cytosolic and mitochondrial aconitase activities in mouse tissue lysates were assayed after cellulose acetate electrophoresis. Note that relative amounts of the two aconitases vary greatly between tissues and that cytosolic aconitase is mainly detected in the kidney, liver and brown fat. (B) Gel retardation assays of IRPs. Lysates from wt tissues (9 μg protein/lane) were incubated with ³²P-IRE and resolved on a 10% nondenaturing gel. Assay without 2-ME reflects IRE-binding activity in vivo (top panel). Assay with 2% 2-ME activates IRP1 that was functionally in a non-IRE-binding state to bind IREs in vitro. A representative example of at least three experiments is shown. In the bottom panel, the small percentage of IRP1 that is in the IRE-binding form is shown as cross-hatched blocks in each column. Notably, 4–18% of IRP1 is in the IRE-binding state prior to 2-ME activation in the seven tissues analyzed. Results shown are representative of three independent experiments.

Steady-state levels of IRP2 increase in the cerebellum and spleen of IRP1−/− mice. (A) Western blots of equal amounts of cerebellar or spleen lysate from two wt (lanes 1,2), IRP2−/− (lanes 3,4) and IRP1−/− (lanes 5,6) mice show a two-fold increase in IRP1−/− animals compared to control. The blot shows duplicates that are representative of four separate experiments from the cerebellum and three separate experiments from the spleen, and results are represented in a bar graph in (B) after quantitation and statistical comparison (P=0.0001 for cerebellum and P=0.035 for spleen).

Targeted deletion of IRP1 in mouse. (A) Schematic diagram of the IRP1 deletion construct (top), IRP1 genomic clone (middle) and IRP1 deletion allele (bottom). (B) Southern analysis of BamHI digest of genomic DNA isolated from mouse tailcuts. In lane 1, wt genomic DNA shows a band of 10.1 kb. In lane 2, genomic DNA from a heterozygote mouse shows a wt band of 10.1 kb and a mutant band of 4.1 kb. In lane 3, DNA from a homozygous mutant shows only a 4.1 kb band. In all three lanes, a wt band for IRP2 is visible at 15.1 kb. (The probe used for IRP1 detection is indicated in (A).) (C) Cytosolic and mitochondrial aconitase activities in mouse liver and kidney lysates were assayed after cellulose acetate electrophoresis. Note that lysates from wt mice show both cytosolic and mitochondrial aconitase activity, while in lysates from IRP1−/− mice cytosolic aconitase activity is absent. (D) Gel retardation assays of IRPs. Lysates of wt and IRP1−/− mouse embryonic fibroblasts (15 μg protein/lane) were incubated with ³²P-IRE and resolved on a 10% nondenaturing gel. Both IRPs respond to decreased iron concentration with increased IRE-binding activity. In lysates from IRP1−/− cells, IRP1 IRE-binding activity is absent (C: control; F: 100 μg/ml ferric ammonium citrate; D: 50 μM deferoxamine).

Ferritin levels are elevated in multiple tissues from IRP2−/− mice, but are elevated only in the kidney and brown fat of IRP1−/− mice. (A) Western blot analysis of ferritin in the liver, kidney and brown fat was performed on tissue lysates of wt, IRP1−/− and IRP2−/− animals using antibodies against mouse liver ferritin. (B) Western blot analysis of ferritin or TfR from the forebrain or cerebellum was performed on tissue lysates of wt, IRP1−/− and IRP2−/− animals using antibodies to mouse ferritin H-chain (forebrain), mouse liver ferritin (cerebellum) or TfR antibodies. The slower running band in forebrain ferritin is L chain that crossreacts with anti-H-chain antibody. Results are presented in duplicate and are representative of at least three experiments.

IRE-binding activity of IRP1 is not recruited by a low-iron diet. Mice were maintained on a low (no iron added)- or high (0.3 g ferric ammonium citrate/kg chow)-iron diet after weaning for a period of 4 months prior to killing. Tissues were prepared, and gel retardation assays and Western blot analysis for ferritin were carried out as described previously. Duplicates are shown of lysates from age-, sex- and diet-matched mice. Note that although IRP1 IRE-binding activity is not recruited by the low-iron diet, IRP2 binding activity increases significantly under the same conditions. Results are presented in duplicate and are representative of at least three experiments.