Fig. 5.

UbcH5-O-Ub is not used as a platform for the synthesis of poly-Ub chains. (A) Reaction scheme depicting how UbcH5c ∼ Ub could conceivably be used, in the absence of a bona fide substrate, to yield UbcH5c ∼ Ub_n via the sequential addition of Ub. (B) Test of the hypothesis. Lanes 1–3: Selective visualization of UbcH5-O-Ub_T7 and its derivatives in the reaction mixture. Equimolar amounts (5 μM) of UbcH5-O-Ub_T7 and wt UbcH5 ∼ Ub were present at the start of the reaction. Samples were collected at 0, 3, and 10 minutes after the addition of SspH2_477–788. The primary products detected are UbcH5-O-Ub₂ and UbcH5-O-Ub₃. Lanes 4–6: Identical reaction mixture except all Ub in the reaction mixture has a T7-epitope tag. The predominant reaction products are poly-Ub chains. A cross-reacting species that is present prior to the addition of SspH2_477–788 is indicated by an asterisk.