Figure 7. USP5 removes the C-terminal extension of UBB.

(a) Human UBB pretreated or not with UCHL3 was incubated with either Otulin (8 ng/lane; upper panel) or USP5 (40 ng/lane; lower panel) for the indicated periods of time. Samples were then subjected to pegylation (+PEGmal), as indicated. Pegylated species (UBB and cleavage products) are marked with asterisks. Note that in the Otulin assay, Ub₂ and Ub also indicate species containing the C-terminal cysteine. (b) Mouse UBB (WT) and UBBY305P (Y305P) were incubated with increasing amounts of USP5 for 30 min at 37 °C (left panel). Processing of UBBY305P with 1 μg of USP5 (lane U5) or 10 ng of Otulin (lane Otl) is also shown (right panel). (c) Mouse UBB pretreated or not with UCHL3, as indicated, was incubated at 37 °C for 30 min with increasing amounts of USP5, as specified. Coomassie blue-stained gels are shown. Numbers to the left indicate the molecular weights of protein standards in kDa.