Fig. 7. Ligand-induced alterations of TRAF-2 localization in living cells. (A,D,E) TNF-R2 signaling was induced in living Hela-TNF-R2 cells using an agonistic TNF-R2-specific antiserum (A). The distribution of TRAF2-YFP was analyzed online both prior to stimulation and for a further 30 minutes after stimulation by confocal microscopy. TRAF2 accumulated in small granular structures in response to TNF-R2 activation, examples of which are indicated with arrows. In contrast, a normal rabbit control serum (B) or the CD30-specific Ki-1 antibody (C) had no effect on the distribution of TRAF2. (D) HeLa-TNF-R2 cells were transfected with a TRAF2-GFP expression plasmid along with pECFP-Mem encoding a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin and CFP. The next day, nonstimulated or TNF-R2-stimulated (1 hour, 2 µg/ml TNF-R2 IgG) living cells were analyzed by confocal microscopy. (E) HeLa-TNF-R2 cells were transfected with a TRAF2-GFP expression plasmid. The following day, nonstimulated, TNF-R2-stimulated (1 hour, 2 µg/ml TNF-R2 IgG) and TNF-treated (1 hour, 20 ng/ml TNF) living cells were analyzed by confocal microscopy. Endogenous TNF-R2 expression was detected by immunofluorescence using a TNF-R2-specific polyclonal rabbit antiserum. Examples of colocalized TNF-R2 and TRAF2-GFP are indicated with arrows.