Figure 5.

H2B-Ub nucleosomes are resistant to Ubp10 DUB activity. A, native gel of reconstituted mononucleosomes containing either unmodified human histone octamer (H2B) or H2B-Ub-assembled octamer. Mononucleosomes were reconstituted via salt dialysis using 147-bp DNA template containing a 601 Widom sequence. DNA–protein complexes were run on a 5% native polyacrylamide gel and stained with SYBR Gold. B, in vitro deubiquitinase assay (DUB assay) was performed using 5 nm recombinant Ubp10 and 100 nm nucleosomal H2A/H2B-Ub or 100 nm free H2A/H2B-Ub heterodimers. Deubiquitination activity was determined by SDS-PAGE followed by Western blot analysis using αH2B and looking at formation of cleaved H2B. Samples were quenched at indicated time points.