Fig. 3.

Autoradiogram of an E2 screen conducted in the presence of Trim25 ligase. Incubations were performed as described in Subheading 3.2 in 10-min incubations containing 100 nM Uba1 and 1 μM recombinant human GST-Trim25 (determined as total protein) in the absence (lane 2) or presence of 100 nM of the indicated recombinant E2 proteins. The concentrations of active E1 and E2 proteins were determined by the stoichiometric formation of ¹²⁵I-ubiquitin thiolester, as described in Subheading 3.1.