Fig. 4.
SspH2 generates poly-Ub chains linked to the active site of UbcH5. Western analysis with α-UbcH5c antibodies performed on a reaction mixture containing purified 37 μM UbcH5-S ∼ Ubwt and 0.6 μM SspH2477–788. Samples were collected at 0, 1, 2, 3, and 4 minutes after addition of SspH2477–788 at 30 °C. Products were separated by SDS–PAGE under nonreducing (-DTT) and reducing (+DTT) conditions. The samples at 0 minutes were collected prior to the addition of SspH2477–788. Incubation at 30 °C of a similar control sample containing only UbcH5-S ∼ Ubwt and lacking SspH2 revealed no changes in the species detected (Fig. S6). Higher molecular weight species of UbcH5 ∼ Ub(n) are observed only in the presence of SspH2477–788 and under nonreducing conditions.