The full-length TRAF3, but not the N-terminal deletion mutant, is depleted in stimulated cells.

(A) Stable KMH2 transfectants expressing the full-length epitope-tagged TRAF3 cDNA (F-TRAF3) were stimulated with human CD40L for the indicated times. The FLAG-tagged protein was immunoprecipitated from total cell extract and analyzed by Western blotting using anti-TRAF3 antibody. The position of F-TRAF3 is indicated by the arrow. The reason for a doublet band representing TRAF3 in anti-FLAG may be due to either alternatively processed exogenous protein or cross-reacting and coprecipitating endogenous TRAF3. (B) Quantification of 2 independent experiments (mean ± SE) performed as in panel A. The ▪ represents the level of the transfected F-TRAF3 protein. (C) Stable KMH2 transfectants expressing the deletion mutant of TRAF3 (δ300TRAF3) were stimulated with human CD40L for the indicated times. Total cell extracts were analyzed by Western blotting using anti-TRAF3 antibody. The position of endogenous TRAF3 (68 kd) is indicated by the heavy arrow and the TRAF3 deletion mutant (δ300TRAF3, ∼ 35 kd) by the thin arrow. (D) Resistance of mutant (δ300TRAF3) TRAF3 to proteolytic degradation, mean ± SE of 3 independent experiments. Here, the ▪ represents the level of the transfected δ300TRAF3, and the ● represents the endogenous, full-length TRAF3. Unstim indicates unstimulated; IP, immunoprecipitated; IB, immunoblotted.