Preparation of reporter gene construct. The human AQP3 promoter region (?990/+8 cool was amplified by PCR using LA Taq polymerase (Takara). The PCR product was cloned into the pCR2.1 vector using the Original TA cloning kit (Invitrogen, Carlsbad, CA). After the sequence was confirmed by automated DNA sequencing analysis (Macrogen, Korea), it was subcloned into the KpnI/XhoI site of the pGL4.17 [luc2/Neo] vector, a promoter-less firefly luciferase plasmid (Promega, Madison, WI).
Transient transfection and luciferase assay. Transient transfections with DNA Angiotensin 1/2 (5-7) were performed with HilyMax reagent (Dojindo, Kumamoto, Japan) according to the manufacturer’s recommendations. Briefly, cells cultured in 12-well plate were incubated with DNA-transfection reagent mixture at 70–80% confluency. Eighteen hours after transfection, cells were serum-starved for 6 h then treated with TNF-α for 24 h, after which cells were harvested for detection of luciferase activity. Luciferase activity was measured using a luminometer (Lumat LB9507, EG&G Berthold) with the Dual luciferase assay kit (Promega). Co-transfection with pGL4.74 [hRluc/TK] (Promega), which expresses Renilla luciferase, was performed to normalize transfection efficiency.
Manage Your Items