Pcmv6 xl6 plasmid

Twenty-four hours prior to transfections, cells were plated in 96-well plates. Cells were transfected with the specific ERBB2 luciferase reporter construct or the backbone vector (100 ng) plus the internal control plasmid pRL-TK (10 ng) using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). In co-transfection assays, cells were transfected with 100 ng of ERBB2 reporter constructs, 10 ng of pRL-TK, and 150 ng of Sp1 expression vector or empty pCMV6-XL6 plasmid (OriGene, Rockville, MD). Twenty-four hours post transfection, cultures were washed once with PBS; cells were lysed in freshly diluted passive lysis buffer (100 μl/well, Promega) by incubating the plates at room temperature on a shaker at 200 rpm for 60 min. Luciferase reporter gene activities were determined with the Dual-Luciferase Reporter Assay System (Promega) as per manufacturer’s instructions. Light intensity was measured in a Synergy HT luminometer equipped with proprietary software for data analysis (BioTek, Winooski, VT). Corrected firefly luciferase activities were normalized to renilla luciferase activities and expressed relative to the averaged activity of the ₋₄₉₉ERBB2 construct which was assigned an arbitrary value of 100.

Twenty four hours prior to transfections, MDCK cells were plated in 24-well plates. Cells were transfected with the specific canine cbr1 luciferase reporter construct or the backbone vector (150 ng) plus the internal control plasmid pRL-TK (15 ng) using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). In co-transfection assays, MDCK cells were transfected with 100 ng of the cbr1 reporter constructs, 15 ng of pRL-TK, and 150 ng of Sp1 expression vector or empty pCMV6-XL6 plasmid (OriGene, Rockville, MD). Twenty four hours post-transfection, cultures were washed once with PBS; cells were lysed in freshly diluted passive lysis buffer (100 μl/well, Promega) by incubating the plates at room temperature on a shaker at 200 rpm for 60 min. Luciferase reporter gene activities were determined with the Dual-Luciferase Reporter Assay System (Promega) per the manufacturer's instructions. Light intensity was measured in a Synergy HT luminometer equipped with proprietary software for data analysis (BioTek, Winooski, VT). Corrected firefly luciferase activities were normalized to renilla luciferase activities and expressed relative to the averaged activity of the _−230/−21cbr1 construct which was assigned an arbitrary value of 100.