Figure 7.

Specificity for Lys-63-linked Ub chain synthesis by WWP1 is a function of chain length. A, anti-T7 western blots showing the starting material used for quantification of Ub chain linkages formed on WBP2-CT^K222 over time by mass spectrometry. The reaction was quenched in 8 m urea at the indicated times, and the ubiquitinated products were purified under denaturing conditions prior to SDS-PAGE. B, quantification of Ub chain linkages using the ubiquitinated material shown in A. Results are represented as a percentage of the sum of all linkages detected. Error bars represent triplicate measurements (±S.D. of the mean). C, ubiquitination of WBP2-CT^K222 was assayed in the presence of wild-type Ub as described in Fig. 4B. The reaction products were probed with linkage-specific antibodies to detect Lys-63-, Lys-48-, and Lys-11-linked chains. D, ubiquitination of WBP2-CT^K222 was assayed in two rounds of reaction to detect a branching activity by WWP1. WBP2-CT^K222 was first modified with a Lys-63-linked Ub₄ chain, such that the substrate was fully converted to its tetraubiquitinated form (lane 2). A molar excess of a lysine-less Ub (0K Ub) was then added to the reaction. The reaction products were quenched at the indicated times as described in A and detected by anti-T7 immunoblotting. The asterisk denotes a cross-reacting species that originates from an aggregate present in the Lys-63-linked Ub₄ preparation.