Prl sv40 renilla reporter vector

Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin at 37 °C in the presence of 5% CO2. The cells were transiently cotransfected with pRL-SV40 Renilla reporter vector (Promega) as an internal control and pGL3-promoter-A or pGL3-promoter-C using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol at 80–90% confluence in a 24-well culture dish. The control plasmid contained the pGL3 promoter, rather than the GRIN2B 3′UTR. This control was cotransfected with the pRL-SV40 Renilla reporter vector as a positive control for luciferase expression. The cells were harvested 24 hours after transfection and the activities of both the firefly luciferase and Renilla luciferase were measured using the Dual-Luciferase Reporter Assay System (Promega) on Berthold LB940 according to the manufacturer’s protocols. The relative firefly luciferase activities were normalized against the Renilla luciferase activities.

Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin at 5% CO2 and 37°C. At 80–90% confluence in a 24-well culture dish, the cells were transiently cotransfected with pRL-SV40 Renilla reporter vector (Promega) and pGL3-promoter-DGA or pGL3-promoter-DGG or pGL3-promoter using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol. Twenty-four hours after transfection, the cells were harvested and the activities of both the firefly luciferase and Renilla luciferase were measured using the Dual-Luciferase Reporter Assay System (Promega) on Berthold LB940 according to the manufacturer’s protocols. Renilla luciferase values were used to normalize cell counts and transfection efficiency.