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. 2014 Jun 5;53(24):4004–4014. doi: 10.1021/bi500072v

Figure 6.

Figure 6

Select noncatalytic backside residues influence the capacity of UbcM2 to synthesize polyUb chains. (A) In vitro ubiquitylation assays conducted at pH 7.4 (left) or pH 8.5 (right) to compare the patterns of [35S]AO7T ubiquitylation between wt UbcM2 and AQVQMM, a six-residue substitution mutant of UbcM2. Reaction mixtures contained either no Ub (lanes 1 and 4) or wt Ub (lanes 2, 3, 5, and 6). (B) Recombinant ubiquitylation assays using nonradiolabeled HA-AO7T were incubated for 90 min and contained either no Ub (lane 1), UbK0 (lane 2), or wt Ub (lanes 3–7), and the indicated forms of UbcM2 (listed above blots). Reaction products were analyzed by Western blotting with the antibody listed at the left of each blot. The migrations of unmodified AO7T and ubiquitylated AO7T [AO7T-(Ub)n] are shown to the right of the blots. The migration of molecular weight markers is indicated to the left of each blot. All assays were repeated a minimum of three independent times.