Fig. 2.

The DR5 agonistic antibody, AMG655, at concentration ranges that suppress invasion (a and b), suppresses TRAF2 polyubiquitination (c), enhances TRAF2 degradation (d and e) and causes delayed inhibiton of JNK signaling (f). a and b, A549 cells were allowed to invade through transwells coated with Matrigel for 48 h in the bottom well containing the indicated concentration of AMG655. Invaded cells at the lower surface were then stained and quantified (a). Under the tested condition, AMG655 minimally affected cell survival (b). c, HEK293T cells were co-transfected with Flag-TRAF2 and HA-Ub for 30 h and then stimulated with different doses of AMG655 as indicated for additional 90 min. The cells were then harvested for preparation of whole-cell protein lysates, IP and subsequent Western blotting (WB) for the indicated proteins. d-f, A549 cells were exposed to 100 ng/ml AMG655 for the given times (d and f). Moreover, A549 cells were pre-exposed to 100 ng/ml AMG655 for 2 h followed by treatment with 10 μg/ml CHX for additional times as indicated (e). After these treatments, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J Software and were normalized to actin. The results were plotted as relative TRAF2 levels compared to those at time 0 of CHX treatment ((e); bottom panel)