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.
