Interaction of Dleu7 with members of the TNFR superfamily, BCMA and TACI, interferes with NF-κB and NFAT trans-activation. (A) HEK293 cells were cotransfected with 50 ng of pNF-κB-Luc reporter or 250 ng of pNFAT-Luc reporter, respectively, and 50 ng of pRL-TK Renilla reporter constructs. In addition, 1.5 μg of CMV5-empty vector or 1.5 μg of CMV5-hDLEU7-HA was used. In addition, 250 ng of CMV6-BCMA and 25 ng of CMV6-TACI (in the case of NF-κB-Luc) and 5 ng of CMV-NFAT, 100 ng of CMV6-BCMA and 5 ng of CMV6-TACI (in the case of NFAT-Luc) 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 and pNFAT-Luc in HEK 293 cells transfected with CMV5-empty vector was set as 1, respectively. Experiments were repeated 3 times in triplicate. Columns indicate the mean; bars, SD. (B) Left panels show HEK 293 cells cotransfected with GLTX5-HA (negative control) and TACI-Myc, or hDLEU7-HA and TACI-Myc constructs. After lysis, immunoprecipitations were carried out using anti-HA antibody, mouse immunoglobulin G, or anti-Myc antibody. Western blotting was carried out as indicated. Right panels show HEK 293 cells cotransfected with BCMA-myc and FHIT-GFP (negative control), or BCMA-Myc and hDLEU7-GFP constructs. After lysis, immunoprecipitations were carried out using anti-GFP antibody, mouse immunoglobulin G, or anti-Myc antibody. Western blotting was carried out as indicated.