Dleu7 inhibits NF-κB–dependent transcription. (A) NIH-3T3 cells were cotransfected with 250 ng of pNF-κB-Luc reporter and 50 ng of pRL-TK Renilla reporter constructs. In addition, 1.5 mg of CMV5-empty vector or 1.5 mg of CMV5-hDLEU7-HA was used. In addition, cells were treated with 50 ng/mL TNF-α for 6, 4, or 2 hours before lysis where indicated. Firefly and Renilla luciferase activities were assayed with the Dual-Luciferase Assay System from Promega, and firefly luciferase activity was normalized to Renilla luciferase activity. The normalized promoter activity of pNF-κB-Luc in NIH-3T3 cells transfected with CMV5-empty vector without TNF-α treatment was set as 1. Experiments were repeated 3 times in triplicate. Columns indicate the mean; bars, SD. (B) HEK 293 cells were cotransfected with 50 ng of pNF-κB-Luc reporter and 50 ng of pRL-TK Renilla reporter constructs. In addition, 1.5 mg of CMV5-empty vector or 1.5 mg of CMV5-hDLEU7-HA was used. In addition, 100 ng of CMV6-CD40, CMV6-TRAF6, or a combination of both were used where indicated. Firefly and Renilla luciferase activities were assayed with the Dual-Luciferase Assay System from Promega, and firefly luciferase activity was normalized to Renilla luciferase activity. The normalized promoter activity of pNF-κB-Luc in HEK 293 cells transfected with CMV5-empty vector was set as 1. Experiments were repeated 3 times in triplicate. Columns indicate the mean; bars, SD.