Pgl4 basic luciferase plasmid

The SK1-luciferase constructs were created by inserting an ~0.5 kb and an ~0.6 kb fragment encompassing the predicted binding site into the pGL4-BASIC-luciferase plasmid (Promega, Tokyo, Japan). The primers used to amplify the DNA fragments were SP1_P1 (forward 5′-GGAACCAGCTCGTGGCCCGG-3′ and reverse 5′-TGCTGGGCACGAAGTTCTGG-3′) and SP1_P2 (forward 5′-AGGCTCAGTGCCCTCCCCGC-3′ and reverse 5′-TGCTGGGCACGAAGTTCTGG-3′). A commercial plasmid containing a CMV-driven Renilla reporter system was used as an internal control (Promega). HK-2 cells were plated in six-well plates at 50–70% confluence and were co-transfected with the pCMV-SP1 construct or with an equimolar amount of the empty pCMV vector and the pGL4-SP1-P1 or pGL4-SP1-P2 construct utilizing Lipofectamine 3000 reagents (ThermoFisher). The media was changed 2 h prior to transfection. After the media was changed, the cells were incubated in 10% DMEM for 24 h. The luciferase assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). Briefly, 100 ml of luciferase substrate was added to 20 ml of lysate, and luciferase activity was measured using an LB940 Multilabel Reader (Berthold Technologies, Bad Wildbad, Germany). Each luciferase assay was performed in triplicate.

The luciferase assay was done as mentioned in our previous reports [46 (link)]. IH9c2 cells plated in 6-well plates at 50%–70% confluence were transfected with either pCMV-CREB construct or an equal amount of empty pCMV vector and either the pGL4-IGF2R_P1 construct utilizing PureFection™ reagents (System Biosciences). The cells were incubated in 10% DMEM for 24 h and the luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Tokyo, Japan). The assay was performed in triplicates. GF2R-luciferase constructs were created by inserting ~1.3 and ~0.7 kb fragments of the predicted EBPRE sequence into the pGL4-BASIC-luciferase plasmid (Promega). The primers used to amplify the DNA fragments were as follows: IGF2R_P1 (forward: 5′-TCTCATCTCGAGCAATGACTAGTCTTCATGTAACAGC-3′ and reverse: 5′-GCCGC-AAAGCTTGAGTCGAAGCTGCAACGG-3′) and A commercial plasmid containing a CMV-driven Renilla reporter system was used as an internal control (Promega).

For promoter assays, the Id promoter was cloned into the pGL4 basic luciferase plasmid (Promega; Madison, WI, USA), and the resulting construct was transfected into MC3T3-E1 cells using Lipofectamine 3000 (Thermo Fisher, CA, USA). Luciferase activity was measured using a luciferase reporter assay system (Promega). These cells were cotransfected with the pCMV-galactosidase reporter plasmid (Promega) and the transfection activity was normalized by measuring the galactosidase activity.