Rr luc 6xcxcr4 renilla luciferase vector

The IGF1R 3′ UTR clone in pMirTarget was obtained from Origene. The IGF1R 3′ UTR reporter was constructed by amplifying the endogenous IGF1R 3′ UTR from the Origene. XhoI and ApaI sites were added to the 5′ and 3′ ends of the fragment during the preceding PCR reaction and cloned into the XhoI and ApaI site on the Rr-luc-6XCXCR4 Renilla luciferase vector (Addgene). To make the IGF1R 3′UTR mutant construct, site-directed mutagenesis was used to delete 99-105, 2619-2626, and 6661-6667 regions, corresponding to the predicted let-7b binding sites. A firefly luciferase vector was used as transfection and normalization control in all luciferase assays. Constructs were sequence verified before being used in experiments.

The PPP1R1C 3′ UTR clone in pMirTarget was obtained from Origene. The PPP1R1C 3′ UTR reporter was constructed by amplifying the endogenous PPP1R1C 3′ UTR from the Origene. XhoI and ApaI sites were added to the 5′ and 3′ ends of the fragment during the preceding PCR reaction and cloned into the XhoI and ApaI site on the Rr-luc-6XCXCR4 Renilla luciferase vector (Addgene). To make the PPP1R1C 3′UTR mutant construct, site-directed mutagenesis was used to delete 2165-2172 region, corresponding to the hsa-miR-182 binding site. A firefly luciferase vector was used as transfection and normalization control in all luciferase assays. The PPP1R1C plasmid encoding the coding sequence was obtained from BioClone Inc. (USA). Constructs were sequence verified before being used in experiments.