FIG. 4.
TRAF2 deficiency results in impaired CD40 signaling in fibroblasts. (A) Expression of TRAF2, CD40, and actin loading control in CD40-transfected immortalized TRAF2+/+ and TRAF2−/− fibroblasts, as determined by immunoblot analysis of total cell lysates. (B) Phosphorylation of p38 and activation of JNK are significantly reduced in CD40L-stimulated but not IL-1-treated TRAF2−/− CD40 fibroblasts. TRAF2+/+ CD40 and TRAF2−/− CD40 fibroblasts were stimulated with CD40L for 20 or 30 min or with murine IL-1 for 20 min or left untreated. Lysates were immunoblotted for the phosphorylated or total p38 or immunoprecipitated with an anti-JNK1 antibody. The anti-JNK1 immunoprecipitates were examined for activity toward c-Jun as a substrate or immunoblotted with a rabbit polyclonal JNK1 antibody. Data are representative of at least four independent experiments for the CD40L and two experiments for the IL-1 treatment. IVK, in vitro kinase assay. (C) Phosphorylation of Akt is impaired in CD40L-stimulated but not PDGF-treated TRAF2−/− CD40 fibroblasts. TRAF2+/+ CD40 and TRAF2−/− CD40 fibroblasts were treated with 0.5 μg/ml CD40L for 20 or 30 min, with 50 ng/ml PDGF, or left untreated as indicated. Lysates were then immunoblotted for the phosphorylated or total Akt. (D) IκBα degradation is diminished in CD40L-stimulated TRAF2−/− CD40 fibroblasts. TRAF2+/+ CD40 and TRAF2−/− CD40 fibroblasts were treated with CD40L or IL-1 or left untreated, as indicated. Lysates were examined for the expression levels of IκBα or, as a control, β-actin. (E) NF-κB binding activity is significantly reduced in CD40L-stimulated TRAF2−/− CD40 fibroblasts. Nuclear proteins were isolated at various time intervals following stimulation with CD40L and examined for binding to a 32P-labeled oligonucleotide probe containing the HIV-LTR NF-κB binding site. At least four independent experiments were performed and gave similar results. Supershift analysis using antibodies against the p65 and p50 NF-κB subunits confirmed that the DNA-bound proteins at 30 min stimulation represent NF-κB (data not shown).