FIGURE 5.

KDM6B 3′ UTR is targeted by miRNAs. KDM6B 3′ UTR-luciferase constructs (pMIR) including targeted binding sites for miR-138, miR-148a, miR-185, and miR-339-5p (A and B), and KDM6B 3′ UTR-luciferase constructs, lacking the miRNA binding sites, pMIRc, were made (D and E). These constructs were co-transfected with cognate pre-miRNA precursors into ND7 cells. pRL null (5 ng/well) was used as a transfection control. Experiments were performed in quadruplicate with each data point in duplicate. After 48 h luciferase activity was assayed using the Dual Luciferase kit from Promega in a 96-well format employing the Vector2 (PerkinElmer Life Sciences) chemiluminescence detector system. Ambion negative control 2, which does not have a miRNA binding sites was used as a control (C). Experiments were performed in triplicate with each data point in duplicate.