TRAF-3 blocks CD40L-induced AP-1 DNA binding activity. (a and b) 18 hours after transfection, HUVECs were incubated with CD40L, and phosphorylation of JNK (a, left panel), c-Jun (a, right panel), and ERK1/2 (b) was detected by Western blot analysis using phosphospecific antibodies. Blots were reprobed with actin or total ERK1/2 to confirm equal loading. Representative blots from three independent experiments are shown. (c) HUVECs were incubated with CD40L for 6 hours or exposed to shear stress for 18 hours. Cells were stained with antibodies against TRAF-2, TRAF-3, or TRAF-5, and secondary FITC-labeled anti-rabbit antibody (left column), followed by counterstaining with DAPI (middle column). The right column shows the overlay of both. n = 3–5. (d) HUVECs or human cardiac microvascular endothelial cells (HMVECs) were incubated with CD40L for 6 hours. Nuclear and cytoplasmic proteins were isolated, and TRAF-3 expression was detected by Western blot analysis. Topoisomerase I was used as a nuclear marker protein. n = 3–5. (e) HUVECs were transfected with TRAF-2, TRAF-3, TRAF-5, or control vector. HUVECs were stimulated with CD40L for 1 hour, and AP-1 DNA binding activity was determined in nuclear extracts by EMSA using a ³²P-labeled AP-1 oligonucleotide probe. n = 3–6. (f) Specificity of binding was examined by competition with 100-fold excess of unlabeled AP-1 oligonucleotides or mutated AP-1 oligonucleotides (mt). For supershift analysis, CD40-stimulated nuclear extracts were preincubated with an AP-1 antibody. n = 3–6.