FIGURE 2.

TRAF3 inhibits LTBR-induced canonical NFκB activation. A, cells were treated with 100 ng/ml agonist LΤΒR antibody (BS-1) for indicated times, and the lysates were analyzed by Western blot for phosphorylated IκBα (Ser-32/Ser-36) and total IκBα. B, samples from BS-1 stimulated DLD-1 cells were also probed for TRAF3 or GAPDH (loading control). C, DLD-1 cells were left untreated, or treated for 10 min and 20 h with 100 ng/ml BS-1, or 20 ng/ml TNFα, and lysates analyzed for TRAF3 and GAPDH (loading control) by Western blot (n.s., nonspecific bands). D, total lysates from untreated DLD-1 and WiDr cells were analyzed by Western blots for TRAF2 and TRAF3, and band intensities were quantified by densitometry. E, cells were mock transfected (Mock), or transfected with either a nonspecific control siRNA (NS), or TRAF3 siRNA for 48 h. The cells were then left untreated (− lanes), or treated (+ lanes) with BS-1 for 10 min, and samples analyzed by Western blots. Blots were probed for different proteins as indicated (n.s., nonspecific band). F, DLD-1 cells were transfected as in C, and left untreated (unstim.) or stimulated with BS-1 for 4 h. RNA was collected and analyzed by real-time qPCR for IP-10 transcripts (unstimulated: ▧; BS-1 treated: ■). Results are normalized to GAPDH transcripts and shown as average + S.D. from quadruplicate samples.