E2F3 is a direct target of miR-34a during granulopoiesis. (A) Schematic representation of miR-34a binding site in the human E2F3 3′UTR. The numbers (+2730 to +2736) represent the nucleotides relative to the termination codon of human E2F3. (B) Conservation of miR-34a binding site in E2F3 3′UTR in human, mouse, and rat genomes. (C) Sequences of predicted miR-34a binding site of E2F3. (D) Luciferase assays in Kasumi-6 cells transfected with E2F3 3′UTR constructs (wild type and mutant) and miR-34a-pCDNA. Bars represent luciferase activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (E) Kasumi-6 cells were transfected with control and miR-34a-pCDNA vectors. Total protein was analyzed by Western blot analysis with anti-E2F3 antibody. Values below the gel image indicate the E2F3 protein levels normalized to β-tubulin. (F) K562-C/EBPα-p42-ER, K562-ER, K562-C/EBPα-p30-ER, and K562-C/EBPα-BRM2-ER cells were induced with ß-estradiol (5μM) for respective time points. Total protein was analyzed by Western blot analysis with anti-E2F3 antibody. Values below the gel image indicate the E2F3 protein levels normalized to β-tubulin.