Fig. 5. Trans-ubiquitylation by hRUL138. MBP-RUL681-1208-FLAG and His-RUL681-1128-His, lacking the RING domain (schematically depicted on the top; RING⁺ and RING^-, respectively) were co-expressed in E. coli and the material immobilized on amylose resin was subjected to ubiquitylation assays with methyl ubiquitin. Alternatively, the separately expressed proteins were analyzed individually or in a mixture (lanes mix). Reactions were separated by SDS-PAGE, blotted and incubated with either anti-His antibody (left panel; RING^- proteins) or with anti-FLAG antibody (right panel; RING⁺ proteins). The asterisk denotes a probable dimer of the RING^- protein.