Journal: Autophagy

Article Title: Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects

doi: 10.1080/15548627.2016.1254852

Figure Lengend Snippet: PARK2 forms a phospho-ubiquitin-rich complex with VDAC. (A) FLAG-PARK2 and control cells were differentially labeled using SILAC. Equal amounts of mitochondria were mixed, solubilized and subjected to anti-FLAG immunoprecipitation, followed by native elution. Complexes from 2 independent experiments (with label switch) were resolved using BN-PAGE. Gel lanes were cut into equal slices, within the range of the 500-kDa complex, and each slice was analyzed by LC-MS. Shown are relative protein abundance ratios (FLAG-PARK2:control). (B) Solubilized mitochondria from FLAG-PARK2 cells treated with CCCP (3 h) were analyzed by 2D BN/SDS-PAGE, followed by western blotting. Asterisk indicates likely ubiquitinated forms of VDAC. (C) Anti-FLAG immunoprecipitations using mitochondria solubilized from FLAG-PARK2-expressing cells, treated with 10 μM CCCP, or with DMSO as a control, for 3 h. Solubilized mitochondria from COX6A1-FLAG-expressing cells were used as a specificity control.26 Eluates were analyzed by SDS-PAGE, followed by western blotting using antiserum for the indicated proteins. Total, 1%; Eluate, 100%. (D) Immunoprecipitated FLAG-PARK2 complexes from CCCP (3 h)-treated cells were analyzed by 2D gel analysis as in (B). Asterisk indicates likely ubiquitinated forms of VDAC. (E) Immunoprecipitated FLAG-PARK2 complexes from mitochondria, after CCCP or DMSO treatment of cells, were incubated with the deubiquitinase USP2, or the buffer control, for 1 h at 37°C. Samples were split and analyzed by BN-PAGE (right panel) and SDS-PAGE (left panel), followed by western blotting with the indicated antibodies. (F) Mitochondria from FLAG-PARK2 cells treated with CCCP for 3 h were solubilized in buffer containing 2% anti-VDAC serum or the respective pre-immune serum (control). Following solubilization, protein complexes were resolved by BN-PAGE analysis (4-13%) and were subsequently immunoblotted. (G) Mitochondria from wild-type cells treated with 10 µM CCCP, or DMSO control, for 3 h were solubilized and analyzed as in (B).

Article Snippet: The deubiquitinase USP2 was purchased from BostonBiochem® (E-506).

Techniques: Labeling, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, SDS Page, Western Blot, Expressing, Two-Dimensional Gel Electrophoresis, Incubation

Journal: Nature chemical biology

Article Title: An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination

doi: 10.1038/nchembio.801

Figure Lengend Snippet: (a) Schematic illustration of human Cdc20. Asterisks indicate lysine residues. (b) Ubiquitination/dissociation of Cdc20 requires lysines in the N-terminal region. In vitro ubiquitination assays were performed with APC re-loaded with double HA-tagged human WT or Cdc20 mutants as described in . (c) Deubiquitination of ubiquitinated Cdc20 increases its affinity for the APC. Supernatant from an in vitro Cdc20 ubiquitination assay containing ubiquitinated Cdc20 was divided into two aliquots and one aliquot was incubated with the catalytic domain of Usp2 (Usp2-CD). Both aliquots were then re-incubated with a fresh batch of high salt-washed APC to assess the binding of Cdc20. (d) Unmodified Cdc20 outcompetes ubiquitinated Cdc20 for binding to the APC. Ubiquitinated Cdc20 (Ub-Cdc20) was generated as in (c) and mixed with reticulocyte lysate containing unmodified Cdc20 at different ratios. The mixture was then incubated with high salt-washed APC and Cdc20 binding was assessed by Western blot. (e) Cdc20 1-164R is more resistant to TAME-induced dissociation than WT Cdc20. APC beads loaded with WT or Cdc20 1-164R were incubated in mitotic extract +/- TAME for 10 min. A fraction of the beads were then treated with Usp2-CD.

Article Snippet: The catalytic domain of Usp2 (Usp2-CD, Boston Biochem, E-506, >95% by HPLC) was added to one aliquot at 500 nM and EDTA (1 mM) and DTT (5 mM) were added to both aliquots.

Techniques: Ubiquitin Proteomics, In Vitro, Incubation, Binding Assay, Generated, Western Blot

Journal: PLoS ONE

Article Title: Regulation of ErbB2 Receptor Status by the Proteasomal DUB POH1

doi: 10.1371/journal.pone.0005544

Figure Lengend Snippet: A, HeLa cells were treated±POH1 siRNA for 48 hours before incubation with 10 µg/ml cycloheximide. Cells were lysed and analysed by immunoblotting with ErbB2 29D8 and Ab20 antibodies, which recognize intracellular and extracellular epitopes of ErbB2 respectively, EGFR, and tubulin antibodies. B, quantitation shows that both EGFR (by antibody 1005) and ErbB2 (by antibodies Ab20 and 29D8) are turned over more rapidly in POH1 knock-down cells (data averaged from 3 experiments). C. HeLa cells were treated with four On Target Plus oligos (POH1) or with oligofectamine alone for 72 hours before lysis with hot lysis buffer. A higher molecular weight ErbB2 “smear” was observed in all 4 knock-down samples. D The high molecular weight smear associated with ErbB2 immuno-reactivity is sensitive to treatment with a deubiquitinase (USP2). HeLa cells were treated with POH1 siRNA or oligofectamine for 48 hours before lysis in the presence of NEM. ErbB2 was immunoprecipitated and treated in vitro with USP2 catalytic domain (100 nM, 8 hours, 37°C). Samples were analyzed by immunoblotting with ErbB2 antibodies targeting extracellular (Ab20) and intracellular (29D8) domains. Note that the smear detected with Ab20 is lost upon USP2 treatment whilst detection with the intracellular domain antibody increases. As a control for USP2 DUB-activity, EGFR was immunoprecipitated from EGF-stimulated (5 min) HeLa cells and treated in vitro with USP2 catalytic domain before SDS-PAGE and western blotting with anti-Ubiquitin.

Article Snippet: Samples with or without USP2 catalytic domain (100 nM; BIOMOL, UW9850) were incubated in a thermoshaker for 8 hours (37°C, 1000 rpm).

Techniques: Incubation, Western Blot, Quantitation Assay, Knockdown, Lysis, Molecular Weight, High Molecular Weight, Immunoprecipitation, In Vitro, Control, Activity Assay, SDS Page, Ubiquitin Proteomics

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet: DDI2 Cleaves Ubiquitylated NRF1 Protein In Vitro (A) Western blot analysis of NRF1 protein, isolated via GST-DSK2 chromatography, after treatment with DDI2, RAD23, and USP2, as indicated (4%–15% TGX). The mobility of NRF1 and cleaved NRF1 is shown on the right (see also arrow for cleaved NRF1). Zoomed-in images of lanes 13 and 14 and 19 and 20 are shown below. Note that large amounts of extract were used as input for GST-DSK2 purification, meaning that in relative terms, ~20× “extract equivalents” were loaded in lanes 5–20 compared to lanes 1–4. See also Figure S6 A. (B) Reprobing of the membrane from (A), with anti-ubiquitin antibodies. For simplicity, only the relevant lanes 5–9 are shown. (C) Experiment as in (A), but testing DDI2 D→N , as indicated. (D) As in (A) and (B), but testing cleavage of exogenously expressed, Myc-tagged versions of NRF1. N, normal NRF1 sequence. m, mutated sequence. Lower panel, lanes 2 and 6 are shown in the enlargement. The “dot” in lane 6 is not a cleavage band. See also independent experiment in Figure S6 B.

Article Snippet: Recombinant Human USP2 Catalytic Domain, CF His tagged , CALTAG Medsystems LTD , Cat# AG-40T-0539-C050.

Techniques: In Vitro, Western Blot, Isolation, Chromatography, Purification, Sequencing

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet: DDI2 Fails to Cleave Purified Ubiquitin Chains but Preferentially Binds and Cleaves Slowly Migrating Ubiquitylated Species (A) Coomassie-stained gels of different commercially available ubiquitin chains before and after incubation with DDI2, RAD23, or the catalytic domain of USP2, as indicated. Untreated substrates are indicated by stippled boxes. Note that USP2 cleaves all these chains to mono- or di-ubiquitin. See also lack of detectable mono-/di-ubiquitin in the blot of Figure S5 D. (B) Schematic of experiments in (C) and (D). (C) Western blot analysis of ubiquitylated proteins in bound and unbound (supernatant) fractions after incubation with chemically inactivated DDI2 protein (note that two biological replicates of the same experiment are shown; lanes 1 and 2/5 and 6 and lanes 3 and 4/7 and 8). Note the specific depletion of slowly migrating ubiquitylated proteins in lanes 1 and 3 and the enrichment of the same in lanes 5 and 7. (D) As in (C), but ubiquitylated proteins on FLAG-DDI2 D→N beads, incubated with DDI2 or DDI2 D→N (non-FLAG tagged to avoid non-specific “displacement” from the beads), as indicated. Note the disappearance of ubiquitylated proteins from beads after incubation with WT DDI2 (above black stippled line, lane 3) and concomitant release of faster-migrating ubiquitylated proteins into the supernatant (below red stippled line, lane 9). See also ImageJ scanning traces below, with position of stippled lines indicated for reference. Please note that the exposure time of the blot on the right is longer than that on the left; only small amounts DDI2-bound material can be eluted. See also Figure S5 .

Article Snippet: Recombinant Human USP2 Catalytic Domain, CF His tagged , CALTAG Medsystems LTD , Cat# AG-40T-0539-C050.

Techniques: Purification, Staining, Incubation, Western Blot

Journal: Molecular Cell

Article Title: DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1

doi: 10.1016/j.molcel.2020.05.035

Figure Lengend Snippet:

Article Snippet: Recombinant Human USP2 Catalytic Domain, CF His tagged , CALTAG Medsystems LTD , Cat# AG-40T-0539-C050.

Techniques: Recombinant, Mass Spectrometry, Western Blot, Sequencing, Plasmid Preparation, Software, Transfection