Fig 2: Characterization of HeLa cells cytosolic DUBs acting on Ub-RPs.(a) SDS-PAGE/Coomassie blue staining analyses of UBA52 and UBA80 processing by HeLa cells cytosolic 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, USP7 and UCHL3 are also shown. The protein standards used to calibrate the column, as well as the void volume (V0), are indicated. (b) Western blot analysis of UCHL3 in HeLa cells transfected with UCHL3-specific siRNA oligos #1 and/or #2, or with a scrambled (sc) control. The corresponding Ponceau S-stained membrane is shown to assess protein loadings. (c) SEC profiles of UBA52-cleavage activity in control (scrambled) and UCHL3 knocked-down (siUCHL3) HeLa cells cytosol. (d) Pooled SEC fractions 4 and 5 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 cleavage (upper panel). The distribution of USP9X in the samples is shown (lower panel). (e) Pooled fractions 6 and 7 (6 + 7) from SEC were incubated with HA-UbVME (lane +) or with HA-Ub (lane -) and subjected to immunoprecipitation using anti-HA-conjugated beads. A Coomassie blue-stained gel is shown. Numbers indicate protein bands of interest that were subjected to mass spectrometry (see Supplementary Table S1). (f) Immunoprecipitation/immunodepletion assay exactly as described in (d) but using pooled SEC fractions 6 and 7, and anti-USP7 IgGs. Lanes I in (a,c,d,f), recombinant UBA52 or UBA80, as indicated. In (a,c–e) numbers to the left indicate the molecular weights of protein standards in kDa.

Fig 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.