Fig. 2
c-Cbl ubiquitinates TRAF6 via lysine 48-linked polyubiquitin conjugation dependent on the Cbl RING finger domain. a – c-Cbl C3AHN, the RING finger mutant, failed to repress TRAF6-mediated NF-κB activation. 293 T cells were transfected with (κB)3-interferon-luciferase reporter plasmid and the indicated combinations of Flag-TRAF6 (0.5 μg), HA-cbl (1 μg), and HA-cbl-C3AHN (1 μg) or pCDNA3.1 (+). The results are the means ± SD of triplicate experiments. b – Controls for ethe xpression of FLAG-TRAF6 and HA-c-Cbl are shown. GAPDH is used as a loading control. c – TRAF6 ubiquitination required the RING domain of c-Cbl. 293 T cells were transfected with the indicated combinations of expression plasmids. After 36 h of transfection, cells were treated with MG132 for 2 h and lysed. Cell lysates were immunoprecipitated with anti-FLAG Ab and then immunoblotted with anti-ubiquitin (ub). Controls for the expression of FLAG-TRAF6 and HA-c-Cbl are shown (right panels). d – 293 T cells were co-transfected with Flag-TRAF6 (1.0 μg), His-Ub (2 μg), and HA-cbl (1.0 μg) as indicated. After 36 h of transfection, cell lysates were immunoprecipitated with anti-FLAG Ab and immunoblottted with anti-ubiquitin. For expression controls, whole cell extracts were subjected to immunoblot analysis with anti-FLAG, anti-HA and anti-ubiquitin as indicated