Renilla tk

Transfection and electroporation experiments were preformed with the following constructs. pmiRZIP lentivector (ZIP NULL), pmiRZIP lentivector anti-miRNA-206 (ZIP-206), and pmiRZIP lentivector anti-miRNA-21(ZIP-21) were acquired from System Bioscience. pMIR206-Luc and pMIR21-Luc were acquired from Signosis BioSignal. Renilla Null and Renilla TK were acquired from Promega (pRL-null number E2271, pRL-TK number E2241).
The mature sequences of miRNA-206 and miRNA-21 were cloned into the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen number K4936-00). The oligos used are shown in Table 2. As negative control the pcDNA6.2-GW/EmGFP-miR-neg control was used, according to manufacturer's instructions.
The 3′UTR of the different analyzed genes was cloned from muscle cDNA into the pMIR-LUC vector (Signosis BioSignal). The sequence of the oligonucleotide primers used to clone the 3′UTR of each gene are shown in Table 2. All constructs were sequenced to confirm the insert and absence of mutation.
To mutate the 3′UTR of YY1 and eIF4E3 the QuikChangeII site-directed Mutagenesis Kit (Stratagene number 200524) was used according to the manufacturer's instructions. The primers used for the mutagenesis are shown in Table 3. All mutated constructs were sequenced to confirm the presence of the desired mutations and the absence of other unspecific mutations.

Cells were transfected with TOPFlash and Renilla-TK (Promega, E2241) plasmids using Lipofectamine 2000 reagent (Invitrogen, 11668019). Forty-eight hours after transfection, the luciferase reporter assay was performed using a Dual Luciferase Reporter Assay System (Promega, E1910). Luminescence data are presented as the firefly luminescence intensity normalized to the Renilla luminescence intensity and then to the control condition.