Figure EV4. Ub hydrophobic patch involvement in linear chain building by the RBR E3 HOIP .

1. Time courses of linear chain building assays using HOIP_{RBR ‐ LDD} and either UbcH5 (left) or UbcH7 (right) conjugated with WT‐Ub or I44A‐Ub as followed by Coomassie‐stained gels. The I44A mutation in the donor Ub does not impair the formation of di‐Ub 9, yet I44A‐Ub significantly reduces linear chain building ability by HOIP_{RBR ‐ LDD}.
2. UbcH7 was pre‐conjugated with either WT‐Ub or mutant Ub (as indicated) and the reaction was quenched with apyrase. Each UbcH7˜Ub species was incubated with H887A‐HOIP_{RBR ‐ LDD} to enable detection of the E3˜Ub intermediate 41. Samples were analyzed on SDS–PAGE under non‐reducing conditions and subsequently visualized by Western blotting for the HA‐tag on Ub. Time was recorded post‐addition of H887A‐HOIP_{RBR ‐ LDD .} Note: The HA antibody cross‐reacts with free HOIP (see *).