text: Transient expression and luciferase assays.
  HEK 293T cells were plated at a density of 5 x 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent (Roche, Basel, Switzerland). Jurkat T cells were plated at 1 x 106 cells/well in six-well culture plates the day of transfection with the appropriate plasmid DNA (~2 mug total) using FuGene 6 transfection reagent. Primary human CD4+ T cells (2 x 106) were nucleofected with the specified plasmid DNA (5 mug total) using the Human T Cell Nucleofection Kit (Amaxa, Gaithersburg, Maryland, United States). Forskolin (10 muM; Calbiochem, San Diego, California, United States) was added in some experiments 20 h posttransfection. Cells were harvested 24 h posttransfection and luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, United States) according to manufacturer's guidelines. pGL4-TKhRluc2 was used as an internal control to normalize for transfection efficiency. Nucleofected CD4+ T cells were also monitored for transfection efficiency and cell viability 24 h posttransfection as follows. Transfection efficiency was routinely ~30% as determined by flow cytometric analysis of EGFP expression. Cell viability, determined by staining with 7-amino-actinomycin D (7-AAD; BD Biosciences), was routinely ~70%. Both transfection efficiency and cell viability in nucleofected CD4+ T cells was independent of the plasmids used. 
  