Fig. 6. (A) HeLa cells were transiently transfected with TNF-R1-YFP and TRADD-CFP. To inhibit the induction of TRADD-dependent apoptosis, cells were treated with z-VAD-fmk (20 µM) immediately after transfection. One day later, transfected cells were analyzed by confocal microscopy. (B) Expression plasmids (120 ng/well) encoding the indicated proteins were transiently transfected into 293 cells along with a 3xNF-B-luciferase reporter plasmid (20 ng/well) and a SV40 promoter-driven ß-galactosidase expression plasmid (10 ng/well). The next day cells were analyzed for luciferase and galactosidase activity. Luciferase activities were normalized according to the respective galctosidase activities. In all transfections z-VAD-fmk (20 µM) was added to block apoptosis. (C) The indicated GFP-tagged variants of TRAF2 were cotransfected along with empty vector or TRADD and cultured overnight in the presence of z-VAD-fmk (20 µM). The next day, the subcellular distribution of the various GFP fusion proteins was analyzed by confocal microscopy. (D-F) The indicated combinations of expression plasmids of TRADD, TRAF2, IKK1-GFP, cIAP2-GFP and cIAP1-GFP were cotransfected and cultured overnight in the presence of z-VAD-fmk (20 µM). The following day, the subcellular distribution of the various GFP fusion proteins was analyzed by confocal microscopy.