E2F1 is a direct target of miR-223. (A) Schematic representation of the E2F1 transcript. Predicted miR-223 binding site is depicted. The numbers (+749 to +754) represent the nucleotides (relative to the E2F1 termination codon) that are predicted to base pair with the miR-223 seed sequence. (B) Sequences of the predicted miRNA-223 binding sites in human, mouse, and rat genomes. Highly conserved nucleotides are shown in gray. (C) Schematic representation of luciferase constructs used for reporter assays. (D) Luciferase assays in 293T cells transfected with vectors shown in panel C and miR-223 and control oligonucleotides (mimic). Bars represent luciferase activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. **P < .01. (E-F) Western blot analysis of E2F1 on whole-cell lysates from sorted Ly6G^pos bone marrow neutrophils from wild-type (WT) and miR-223 knockout (KO) animals¹¹  (E), on whole-cell lysates from U937 cells treated with 20 nm of control and miR-223 oligonucleotides (mimic; F).