Fig. 3.

Inducible binding of TRAF2 and TRAF3 to chromatin and the NF-kB consensus element. a Western blot analysis of the binding of TRAFs 2 and 3 to chromatin in Neuro2a cells, following CD40L stimulation. Neuro2a cells were either untreated (control) or stimulated for 5, 10, or 30 min with mouse CD40L (100 ng/mL), chromatin extracts were then prepared, and a Western blot was performed. Histone H3 was used as a loading control and remained constant in all four conditions. b EMSA analysis of NF-kB DNA-binding activity in Neuro2a extracts following CD40L stimulation, and the effect of TRAF 2 or 3 antibody preincubation on such activity. Neuro2a cells were either untreated (control) or stimulated for 5, 10, or 30 min with mouse CD40L (100 ng/mL), nuclear extracts were then prepared, and an EMSA was performed. The extracts (5 μg) were then either untreated or preincubated with antibody to TRAF 2 or 3, or with 100-fold excess of unlabeled NF-kB or NFAT consensus elements, as controls for the specificity of the NF-kB DNA-binding complexes. Incubation with a 100-fold excess of unlabelled NF-kB promoter element greatly attenuated the DNA-binding of all four bands, while incubation with an excess of unlabeled NFAT element had no effect on any of the four bands, demonstrating that all four bands were specific. c Western blot analysis of the binding of TRAF2, TRAF3, and RelA to the NF-kB consensus element following NF-kB oligoprecipitation performed on CD40L-stimulated Neuro2a nuclear extracts. Neuro2a cells were either untreated (control) or stimulated for 5, 10, or 30 min with mouse CD40L (100 ng/mL), and nuclear extracts were prepared. The extracts (100 μg) were then incubated overnight with NF-kB-cross-linked agarose beads in DNA-binding buffer. The following day, the beads were precipitated and washed and a Western blot was performed. d Western blot analysis of the binding of TRAFs 2 and 3 to chromatin in Daudi cells, following CD40L stimulation. Daudi cells were either untreated (control) or stimulated for 5, 10, or 30 min with human CD40L (100 ng/mL), chromatin extracts were then prepared, and a Western blot was performed. Histone H3 was used as a loading control. C control, T2 TRAF2, T3 TRAF3, Ab antibody