Liver iron loading and duodenal protein expression in mice with Cybrd1 mutations. (A) There was slightly less nonheme liver iron content, on average, in Cybrd1^-/- animals compared with wild-type animals (12-week-old males: wild type [n = 3], Cybrd1^-/- [n = 3]; 4-week-old females: wild type [n = 9], Cybrd1^-/- [n = 7]; 12-week-old females: wild type [n = 10], Cybrd1^-/- [n = 9]). The differences did not reach statistical significance, but the trend was greatest in female mice maintained on an Fe(-) (iron deficient) diet. Values are shown as a percentage of the values seen in wild-type mice. (B) Liver nonheme iron content (calculated for whole liver and adjusted for body weight) in 12-week-old female mice fed an iron-deficient diet for 8 weeks (n = 9-10). Results in μg/g wet weight are expressed as mean ± SEM. Statistical analysis was performed using the unpaired Student t test, comparing mutant versus wild-type mice, P = .019. (C-D) Immunoblot analysis of duodenal lysates from Trf^hpx/hpx (lanes 1 and 7), iron-deficient wild-type (lanes 2-3), and iron-deficient Cybrd1^-/- (lanes 4-5) mice using anti-Cybrd1 antiserum (C) or anti-Slc11a2 antiserum (D). The single band shown in panel D ran between the 60-kDa and 85-kDa markers. No sample was loaded in lane 6. Protein amounts loaded per lane were 10 μg wild-type and Cybrd1^-/- lysate, and 2 μg (lane 1) and 4 μg (lane 7) Trf^hpx/hpx duodenal lysate. (E) Immunoblot analysis of duodenal lysates from Trf^hpx/hpx mice (lanes 1-2), a Cybrd1^-/- mouse maintained on an iron-deficient diet for 27 weeks (lane 3), a wild-type mouse maintained on an iron-deficient diet for 27 weeks (lane 4), and a wild-type mouse on a regular iron diet (lane 5) using anti-Slc11a2 antiserum, anti-Cybrd1 antibody, or anti-Slc40a1 antibody.