Fig. 3
CC domain of TRAF6 is required for processive polyUb chain synthesis. a Schematic diagram of FL TRAF6 and its deletion mutants used in this study. b Deletion of CC domain (ΔCC) decreases polyUb chain assembly. Flag-tagged TRAF6 or the indicated deletion mutants were expressed in 293T/TRAF6 KO cells and purified using anti-Flag M2 magnetic beads and then used in in vitro ubiquitination assay. c, d Deletion of CC domain (ΔCC) decreases the rate of polyUb chain assembly. In vitro ubiquitination assay was carried out to compare time-dependent polyUb chain assembly between TRAF6 FL and ΔCC. Reaction aliquots quenched at the indicated times were evaluated by SDS-PAGE and immunoblotting (c), which were quantified and plotted as function of time in d. R ob, arbitrary observed rate of polyUb assembly. e CC domain is required for NF-κB activation by TRAF6 in cells. As in Fig. 2d, but TRAF6 or its deletion mutants were used. The data are showed as the mean ± s.d. of triplicate independent sets of experiments. f CHIP-Z3CC activates NF-κB. Upper panel, diagram of CHIP-Z3CC fusion. Z3CC: Zinc fingers 3, 4 and CC domain of TRAF6. Bottom panel, as in Fig. 2d, reporter assays were performed with TRAF6, CHIP, Z3CC, and CHIP-Z3CC to assess their ability to activate NF-κB. The data are showed as the mean ± s.d. of triplicate independent sets of experiments
