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. 2015 Aug 3;5:12836. doi: 10.1038/srep12836

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(a) SDS-PAGE/Coomassie blue staining analysis of UBB processing by SEC fractions (upper panel). Lane C, total cytosolic proteins were also assayed. The elution profiles of USP5 (middle panel) and of the protein recognized by the anti-Otulin antibody (lower panel) are also shown. The elution positions of molecular mass protein standards, as well as the void volume (V₀), are indicated. (b) The UBB cleavage activity comprises an HA-UbVME-sensitive and an HA-UbVME-insensitive component. SEC fractions 7–12 were preincubated in the absence or presence of HA-UbVME or HA-Ubal and used in UBB processing activity assays. Inhib, HA-UbVME or HA-Ubal. (c) SEC fraction 8 was subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP5 IgGs (lanes USP5). Fraction 8 (F8) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for processing activity (upper panel). The distribution of USP5 in the samples is shown (lower panel). (d) Recombinant Otulin (10 ng) pretreated or not with HA-UbVME or HA-Ubal, as indicated, was incubated with UBB for 10 min at 37 °C. A Coomassie blue-stained gel is shown. Cleavage intermediates (Ub₃ and Ub₂) and ubiquitin (Ub) are indicated. Lane I, recombinant mouse UBB used in the assays. Numbers to the left indicate the molecular weights of protein standards in kDa.