Fig. 3. Receptor-induced depletion of cytoplasmic TRAF2. (A) Cell lysate (200 µl) of untreated HeLa-TNF-R2 cells were separated by size exclusion chromatography on a HR10/30 Superdex 200 column and the fractions (0.5 ml) were analyzed by immunoblotting with a polyclonal TRAF2-specifc rabbit antiserum. Elution volumes of molecular mass standards are indicated above. (B,C) HeLa-TNF-R2 cells (B) and HeLa cells expressing a deletion mutant of TNF-R2 lacking the entire cytoplasmic domain (C) were stimulated for 6 hours with an agonistic TNF-R2-specific rabbit IgG fraction or remained untreated. Cell lysates (200 µl) derived from these groups were separated by size exclusion chromatography and corresponding fractions of stimulated (+) and unstimulated (-) cells were compared with respect to TRAF2 recovery by immunoblotting. (D,E) HeLa-CD40 (D) and HeLa-CD30 (E) cells were stimulated for 6 hours with crosslinked soluble CD40L (100 ng/ml) and the CD30-specific mAb Ki-1 (3 µg/ml), respectively, or remained untreated. The lysates were then analyzed as described in B and C. (F) Lysates from HeLa-TNF-R2 cells stimulated for the indicated times with TNF-R2-specific IgG (2 µg/ml) were separated by gel filtration and the TRAF2 content of corresponding fractions were compared by immunoblotting. (G) HeLa-TNF-R2 cells were stimulated for the indicated times with TNF-R2-specific IgG (2 µg/ml). Total cellular content, soluble proteins and the detergent-insoluble compartment (DIC) fractions were prepared as described in Materials and Methods and analyzed with respect to TRAF2 by western blotting. (H) To thoroughly quantify the loss in the total TRAF2 content observed in receptor-stimulated cells after 18 hours, six independent experiments were analyzed. Cells were stimulated for 0, 3 and 18 hours, boiled in SDS-PAGE sample buffer and analyzed by western blotting.