Fig. 4
CC domain enables processive polyUb chain synthesis by TRAF6. a, b Processive polyUb chain assembly by TRAF6. Time-course analysis of polyUb chain synthesis by T6RZC was performed as indicated and shown in a. The corresponding quantification was plotted as function of time in b. R ob, arbitrary observed rate of polyUb assembly. c, d Lack of CC impairs polyUb chain synthesis by TRAF6. Time-course analysis of polyUb chain synthesis was performed to compare T6RZC and T6RZ. Time-dependent accumulation of polyUb chains was shown in c and quantified in d. It is needed to note we used a lot more of T6RZ in order to enhance the rate by T6RZ so we could visualize the products and do the reliable quantification. R ob, arbitrary observed rate of polyUb assembly. e–g Km determination of Ub~Ubc13 for T6RZC and T6RZ. Pre-charged Ub~Ubc13/Uev2 were titrated into T6RZC or T6RZ and incubated at 30 °C for 30 min. The products were immunoblotted (e, f), quantified and plotted as function of Ub~Ubc13/Uev2 in g. The Km values were estimated by fitting two independent data sets to Michaelis–Menten kinetics
