miR-451 represses Cox10 in FL erythroblasts. (A) Ago HITS-CLIP signals in the 3′ UTRs of Cab39 (a known miR-451 target) and Cox10 mRNAs in WT and miR-144/451 KO erythroblasts. The RNA sequences are shown with canonical seed matches in red. (B) Expression of Cab39 and Cox10 spliced mRNA and primary transcripts in WT and KO FL erythroblasts. (C) Western blot showing Cox10 protein expression in WT and KO FL erythroblasts (3 biological replicates). Bar graphs indicate relative expression normalized to β-actin. (D) Cox10 mRNA quantified by qRT-PCR in WT and KO FL erythroblasts following transduction with retroviral vectors expressing control, miR-144, or miR-451. (E) Firefly luciferase activity normalized to Renilla luciferase activity in 3′ UTR reporter assay. 293T cells were transfected with plasmid vectors encoding a Firefly luciferase reporter fused to the 3′ UTR of Cox10 or Cab39 mRNA along with constitutively active plasmid vectors encoding miR-451 or a control nontargeting microRNA and Renilla luciferase. Luciferase activity was measured at 48 hours. (F) Western blot showing Cox1 protein in WT and KO FL erythroblasts. Cox1 is a mitochondrial complex IV protein that is stabilized by Cox10. Numbers below indicate relative expression normalized to β-actin. Bar graphs show mean plus or minus SEM. *P < .05, **P < .01, ***P < .001.