Luciferase reporter pmir control vector

We constructed sensor vectors by joining the region with or without a possible binding site from the 3′-UTR of human DR5 (No.4271–4650) with a luciferase reporter pMIR-control vector (Ambion, Foster City, CA, USA) to examine the target sequence of miR-133b. To generate sensor vectors with 4 mutations in the binding site of the 3′-UTR of human DR5 (No.4455–4463) for miR-133b, we mutated seed regions from GGACCAAA to GGCATGAA (mt-DR5, PrimeSTAR^® Mutagenesis Basal Kit; TaKaRa). The sensor vector with these mutations was submitted to Life Science Research Center, Gifu University, for DNA sequencing. The cells were seeded in 12-well plates at a concentration of 0.5 × 10⁵/well the day before the transfection. The sensor vector (concentration; 0.5 μg/well) and 10 nM miR-133b or nonspecific control miRNA (Dharmacon) were used for the co-transfection of the cells by using Lipofectamine RNAiMAX (Invitrogen). Forty-eight hours after the co-transfection, luciferase activities were measured by using a Dual-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.