Pgl4.45 luc2p isre hygro

Cells at 40% to 50% confluence in the 12-well plates were transfected with a reporter luciferase plasmid containing the interferon stimulated response element (ISRE) (pGL4.45[luc2P/ISRE/Hygro]; Promega) and containing four tandem copies of the STAT6 binding site (p4xSTAT6- Luc2P; Addgene, Cambridge, MA, USA). The reporter vector was then transfected into the cell lines with Lipofectamine plus reagent (Lifetechnology) according to the manufacturer's instructions. The total amount of plasmid DNA per well was adjusted to be the same by adding suitable amounts of empty vector. To activate the STAT1 and STAT6 reporter vectors, IL-4 (10 ng/ml) and IFN-γ (10 ng/ml) were used to treat to the cells for 6 h. Cells were harvested 48 h after transfection, and luciferase activity was measured using a commercial luciferase assay kit (Promega) on the TD20/20 Turner luminometer (Turner Design Instruments, Sunnyvale, CA, USA). The luciferase activity of each sample was normalized to that of the corresponding sample transfected with pGL4. All experimental and control groups contained at least three wells, and the results were reported as mean absorption±standard error.