Posttranscriptional reduction of ferrochelatase (FECH) activity and protein levels in erythropoietic tissues of IRP2^−/− mice. (A,D) FECH activity in bone marrow aspirates and whole spleens of Irp1^+/+:Irp2^−/− mice was significantly decreased compared with Irp1^+/+:Irp2^+/+ (wild-type) animals. Error bars represent SD (n = 4 animals per genotype). (B,E) FECH protein levels in bone marrow and spleen were also decreased in Irp1^+/+:Irp2^−/− animals. Each lane from the Western blot represents whole-tissue protein extracts pooled from 2 animals. The filter was reprobed for SOD2 as a loading control for mitochondrial matrix protein. (C,F) FECH mRNA levels were increased in the spleens of Irp1^+/+:Irp2^−/− mice. Messenger RNA levels were measured by Northern blot using a probe specific for both the 2.2-kb and 2.9-kb FECH transcripts; each lane represents RNA pooled equally from 2 animals. The 18S ribosomal band was visualized to assess equal loading. Results were confirmed by quantitative RT-PCR (data not shown). (G-H) To estimate the tissue distribution of erythroid cells, β-globin mRNA levels in bone marrow aspirates and spleens from Irp1^+/+: Irp2^+/+, Irp1^+/+:Irp2^−/−, and Irp1^+/−:Irp2^−/− mice were assessed by Northern blot. (I) FECH protein abundance in splenic TER119⁺ erythroid cells mice was measured by Western blot. FECH mRNA transcript levels (J, ■) were measured by quantitative RT-PCR using primers specific for both the 2.2-kb and 2.9-kb FECH transcripts; data are normalized to Irp1^+/+:Irp2^+/+ (wild-type) mRNA levels. Relative protein abundance was calculated by densitometry, and individual protein abundance values were normalized to the respective mRNA levels measured in the same sample (J, □). Data in panels A, D, and J were analyzed by 2-tailed Student t test; *P < .05; **P < .01.