Generation of TRAF3 transgenic mice. (A) Representation of the TRAF3 transgenic constructs. The various components of transgenic cassette are indicated. (B) Immunoblot analysis of the hTRAF3 transgene expression in various tissues from the TRAF3 transgenic mice (ϵ-line) and wild-type littermates. hTRAF3 expression was detected using an antibody that specifically recognizes human TRAF3, but not mouse TRAF3.16 (C) Analysis of TRAF3 and IκBα expression in total splenocytes and in affinity-purified B lymphocytes and T lymphocytes isolated from TRAF3 transgenic mice and wild-type littermates. The TRAF3 antibody used recognizes only human TRAF3.16 The purity of B cells and T cells was greater than 95%. (D) Purified B cells from spleens of the various transgenic mice indicated and wild-type littermates were lysed in Laemmli buffer and sonicated. Lysates were normalized for protein content, and 25 μg were immunoblotted. Total TRAF3 and IκBα levels are shown. The TRAF3 antibody used (C-20) recognizes both human and mouse TRAF3. (E) B cells from wild-type mice were left resting or stimulated with 2 μg/mL LPS for 16 or 72 hours. TRAF3 and Bcl-2 expression were analyzed by immunoblotting. (F) Kaplan-Meier analysis of the lifespan of wild-type and TRAF3 littermates (17 mice per group). Statistical significance was calculated by log-rank analysis. (G) The weight of spleens from mice up to 8 months (TRAF3, n = 7; wild-type, n = 5) or more than 16 months old (TRAF3, n = 27; wild-type, n = 19) was determined. Statistical significance was determined by unpaired Student t test.
