Reporter gene assays.

HEK293T, Jurkat, and HepG2 cells were transfected using the Lipofectamine 2000 transfection reagent according to the manufacturer's instructions. Each transfection experiment was performed at least in triplicate; data are presented as the means with standard deviations. In general, HEK293T cells were transfected in a 6-well dish with 20 ng TK-renilla (the transfection control), 200 ng a luciferase construct (IRF-1–luc or κB-luc), limiting amounts of p65 (50 ng) or IRF-1 (25 ng), and titrating amounts of FLAG-HBZ, FLAG–APH-2, or the control expression vector (approximately 2,000 ng total DNA per well). Jurkat cells were transfected in a 6-well dish with 200 ng TK-renilla (the transfection control), 500 ng the luciferase construct (κB-luc), 500 ng p65, and titrating amounts of FLAG-HBZ, FLAG–APH-2, or the control expression vector (approximately 4,000 ng total DNA per well). HepG2 cells were transfected in a 6-well dish with 200 ng TK-renilla (the transfection control), 500 ng the luciferase construct (9×CAGA-luc), and titrating amounts of FLAG-HBZ, FLAG–APH-2, or the control expression vector (approximately 2,000 ng total DNA per well). At 24 h posttransfection, exogenous TGF-β (10 ng/ml; R&D Systems, Minneapolis, MN) was added to the HepG2 cell culture medium. HEK293T cells were harvested at 24 h posttransfection, and Jurkat and HepG2 cells were harvested at 48 h posttransfection. The cells were placed in passive lysis buffer (Promega, Madison, WI). The relative amounts of firefly and renilla luciferase were measured by a FilterMax F5 multimode microplate reader using a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Assays were performed under each condition in duplicate. Extracts were also subjected to immunoblotting to verify the presence of equivalent protein levels.