Fig. 4. Self-ubiquitylation of hRUL138. (A) Polyubiquitylation of full-length hRUL138. hRUL138 was in vitro translated in the presence of ³⁵S-Met and incubated with ATP and E1 plus ubiquitin (Ub) and UbcH5 or UbcH7 as indicated, and the reaction products were analyzed by SDS-PAGE and autoradiography. The borders of the stacking gel are indicated; material accumulating at the upper edge of the stacking gel is marked poly-Ub RUL. (B) Effect of methyl ubiquitin. hRUL138, and the RING containing fragment 681-1208 were in vitro translated and processed as in A, except that methyl ubiquitin (MeUb) was used in two reactions. Both proteins were efficiently modified except that the reaction products were smaller (smear extending to the top of the separating gel; lanes 9 and 12). (C) Requirement for an intact RING-H2 domain. RUL681-1208 and its C1187S mutant were in vitro translated and subjected to ubiquitylation assays as in A.