Time course of CD40-mediated activation of NF-κB and depletion of TRAF3 in KMH2 cells.

KMH2 cells were stimulated with human CD40L for the indicated times. (A) Nuclear extracts were prepared, followed by EMSA. One microgram of each extract was incubated with radiolabeled double-stranded oligonucleotide containing the NF-κB DNA binding motif. Binding reaction was performed either without a competitor or in the presence of 30-fold excess of unlabeled competitor oligonucleotide (comp) containing wild-type NF-κB binding site (wild-type, wt), mutant NF-κB binding site (mt) or a nonspecific sequence estrogen response element (ns). The arrowhead indicates the position of the protein-DNA complex. (B-D) The level of TRAF3 was detected by Western blot using antihuman TRAF3 antibody in total cell (B), membrane (C), and cytoplasmic extracts (D). The position of TRAF3 (67 kd) is indicated by an arrow. In panels B and C, the level of the “housekeeping” enzyme GAPDH is detected to demonstrate equal protein content in each lane. In panel D, the membrane-bound CD30 is used for this purpose. The results are representative of 3 independently performed experiments.