NOTCH1 is a direct target of miR-30c. (A) Schematic representation of miR-30c site in the human NOTCH1 3′UTR. The numbers (+1520 - +1526) represent the nucleotides relative to the termination codon of human NOTCH1. (B) Conservation of the miR-30c binding site in the NOTCH1 3′UTR of human, mice, rat, and dog. (C) K562-C/EBPα-p42ER and (D) K562-C/EBPα-p30ER cells were induced with β-estradiol (5 µM) for respective time points. Total protein was analyzed by western blot analysis with the anti-Notch1 antibody. Values below the gel image indicate the Notch1 protein levels normalized to GAPDH. Total RNA was analyzed by Q-RT-PCR with oligonucleotides for Notch1. Data are represented as mean ± SD from 3 independent experiments. (E) K562-C/EBPα-p42ER cells were induced with β-estradiol (5 µM) for 6 hours. Total protein was analyzed by western blot analysis with anti-Trib2 antibody. Values below the gel image indicate the Trib2 protein levels normalized to GAPDH. (F) K562 cells were transfected with control and pcDNA6.2-GW/EmGFP-miR-30c vectors and cultivated for 48 hours. Total protein was analyzed by western blot analysis with the anti-Notch1 antibody. Values below the gel image indicate the Notch1 protein levels normalized to GAPDH. (G) Hematopoietic CD34⁺ cells were cultured as discussed in “Methods.” Total protein was analyzed by western blot analysis with the anti-Notch1 antibody. Values below the gel image indicate the Notch1 protein levels normalized to GAPDH. (H) Schematic representation of predicted and mutated miR-30c binding site in the luciferase construct used for reporter assays. (I) Luciferase assays in MV4-11 cells cotransfected with Notch1 3′UTR constructs (wild-type and mutant) and pcDNA6.2-GW/EmGFP-miR-30c. The bars represent luciferase activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. **P ≤ .01.