Figure 3. Characterization of mouse liver cytosolic DUBs acting on Ub-RPs.

(a) SEC profile of HA-UbVME-reactive DUBs from mouse liver cytosol. Total cytosolic proteins (lane C) and the corresponding SEC fractions (lanes 1–16) were reacted with HA-UbVME and analyzed by SDS-PAGE/western blotting with an anti-HA antibody. Arrows a–c indicate HA-UbVME-reactive DUBs that co-elute with enzyme activities described in the main text. The elution positions of molecular mass protein standards, as well as the void volume (V₀), are indicated. (b) SDS-PAGE/Coomassie blue staining analyses of UBA52 and UBA80 processing by SEC fractions (first and second panels, respectively). Lane C, total cytosolic proteins were also assayed. Ubiquitin (Ub) and the ribosomal proteins L40 and S27A are indicated. The elution profiles of USP9X and UCHL3 are also shown (lower panels). (c) Pooled fractions 4 and 5 from SEC were subjected to an immunoprecipitation/immunodepletion assay using control (lanes Ctrl) or anti-USP9X IgGs (lanes USP9X). Pooled fractions (4+5) and the corresponding immunoprecipitated (lanes IP) and immunodepleted fractions (lanes ID) were assayed for UBA52 and UBA80 cleavage (upper and middle panels, respectively). The distribution of USP9X in the samples is also shown (lower panel). Lanes I, recombinant Ub-RP fusion proteins used in the assays. Numbers to the left indicate the molecular weights of protein standards in kDa.