Trf-LKO mice have impaired iron metabolism. (A) Hepatic Trf protein and serum Trf levels were measured in 8-week-old control (Trf^fl/fl) and Trf-LKO (n = 6-7 mice/group) mice. (B) Serum iron levels, serum NTBI levels, and tissue non-heme iron concentrations were measured in 8-week-old control and Trf-LKO mice (n = 8-10 mice/group). (C) Hepatic Tfr1, Ftl, and Fth mRNA levels were measured in control and Trf-LKO mice using RT-PCR (n = 6 mice/group). (D) Western blot analysis of hepatic Tfr1, ferritin-L (Ftl), and ferritin-H (Fth) proteins in control and Trf-LKO mice (n = 3 mice/group). (E) RT-PCR analysis of hepatic Hamp mRNA in control and Trf-LKO mice (n = 6 mice/group). (F) Perls Prussian blue staining for iron and Fpn1 IHC were performed in liver, spleen, and duodenum sections obtained from control and Trf-LKO mice; the scale bars represent 100 μm. *P < .05, **P <. 01, Student t test. n.s., not significant.