Prl tk renilla reniformis luciferase plasmid

For promoter assay, we used pGL3-Enhancer vector (Promega). Indicated promoters were cloned from genome obtained from normal mouse heart, and the sequences were inserted into the upstream of luciferase in pGL3-Enhancer vector. For mutagenesis of the promoters, we used QuikChange Site-Directed Mutagenesis kit (Agilent Technologies) in accordance with the manufacture’s protocol. pRL-TK™ Renilla reniformis luciferase plasmid (Promega) was used as the transfection efficiency control^{32 (link)}.

The promoters of mouse matrix metallopeptidase (MMP)-2, MMP-8, and MMP-9 were amplified and inserted into the pGL3 basic vector. TM4 cells were cultured in 24-well plates and electrotransfected with pGL3 carrying the promotors of MMP-2, MMP-8, or MMP-9 (0.03 μg) and the pGL3-NF-κB plasmid (0.3 μg) using the Neon® Transfection System (pulse voltage: 1700 V, pulse width: 20 ms). Detection was performed using a kit according to the manufacturer’s instructions (Promega).
pMIR-REPORT™ vector was provided by Invitrogen. The pRL-TK™ Renilla reniformis luciferase plasmid was purchased from Promega. To confirm the regulation of Cab39 by miR-451, Cab39′-UTR was also inserted into the 3′-UTR of the pMIR-REPORT™ vector. After that, TM4 cells were cultured in 24-well plates and then electrotransfected with the pRL-TK™ Renilla reniformis luciferase plasmid and pMIR-REPORT™ vector.