Figure 7.

Analysis of the elongation of Ub chains. A and B, determination of chain length distribution. Reaction products obtained under standard conditions for 3, 5, and 7 min (A) or with 4-fold lower E2 concentrations for 10, 20, and 30 min (B) were digested with ^hisUSP7 for the indicated times. C and D, the signal intensities of the respective chains at 20 min of digestion for A and B were divided by the number of Ub molecules in the chains, and the calculated values (relative molar amounts of the respective chains) were plotted in C and D, respectively. E, two-step chain elongation reaction. The time course performed with a low amount of Ub (7.5 pmol) was followed by the addition of a large excess of ^FLAGUb (174 pmol). F, generation of Ub-^FLAGUb hybrid chains. Reaction products obtained under the standard ubiquitination condition for 7 min with Ub, ^FLAGUb, or a mixture of both (lanes 1–3) were digested with USP7 for 80 min (lanes 4–6). G, evidence of stepwise chain elongation. Reaction products before (5-min incubation with Ub, lane 6 in E) and after the addition of ^FLAGUb (additional 3-min incubation, lane 10 in E) were digested with ^hisUSP7 for the indicated times. The product of the reaction performed with Ub and ^FLAGUb (lane 5 in F) was loaded on the side as a standard (S, lane 11). F-Ub, ^FLAGUb. Reaction products were visualized by Western blotting with the indicated antibodies.