Journal: Scientific Reports

Article Title: Expression and Regulation of Deubiquitinase-Resistant, Unanchored Ubiquitin Chains in Drosophila

doi: 10.1038/s41598-018-26364-x

Figure Lengend Snippet: Non-cleavable poly-Ub in Drosophila . ( A ) Summary of poly-Ub constructs. ( B ) Recombinant, untagged Ub 6 (1 µM) was incubated with USP5 (50 nM). See Methods for details. ( C ) Western blots of Ub 6 -Stop expressed in all tissues. Driver was sqh-Gal4. HMW: higher molecular weight. Asterisk: non-specific band. Flies were one day old. ( D ) Western blots from whole flies expressing the indicated transgenes, driven everywhere by sqh-Gal4. HIS 6 -pulldown of Ub 6 -Stop was conducted under denaturing conditions. Solid line separates blots from the same membrane probed with the indicated antibodies. Dotted lines separate lanes reorganized from the same membrane. Un-cropped blots are in Supplemental Figures. ( E ) Histograms summarizing Ub 6 -Stop that is unmodified (non-conjugated band) vs. modified (conjugated bands and upper smear) in blots in panels ( C , F , G ) and other, similar experiments. N = 10 independent repeats. Means −/+SD. Driver: sqh-Gal4. ( F ) Deubiquitination reaction of Ub 6 -Stop isolated from flies and incubated in the absence or presence of the catalytic domain of recombinant USP2 (USP2 CD ). See Methods for details. ( G ) Western blots from denature/renature IPs to examine ubiquitination of HA-tagged Ub 6 -Stop by the indicated linkages. Solid lines indicate that membrane was loaded multiple times with the same samples, same amounts, cut and probed simultaneously, as shown, to eliminate cross-contamination issues from stripping and re-probing the same membrane. Asterisks: non-specific bands that we observe consistently with the respective antibodies. Dotted lines separate lanes cropped from the same membrane. Un-cropped blots are shown in Supplemental Figures. ( H ) Western blots of whole, adult flies expressing Ub 6 -Stop in all tissues (driver was sqh-Gal4), and loaded to probe all Ub species. HMW: higher molecular weight. Box on the right summarizes quantification of signal from the adjacent blots and other, independent experiments, normalized to respective loading controls, with Ctrl. lanes set to 100%. P value is from two-tailed Student’s t-test. Signal quantified encompassed the band at the arrow all the way at the top of the gel. I) Western blots of stringent purification of HA-Ub 6 -Stop driven by tubulin-Gal4-GS, which is dependent on RU486 to induce expression of Ub 6 -Stop in all tissues; see main text for details. Arrows: unmodified Ub 6 -Stop. Curved lines: higher molecular weight species consistent with ubiquitinated Ub 6 -Stop. For panels (B–D,F–I), results are representative of experiments conducted independently at least thrice, with similar results. In all panels with Drosophila data, flies were heterozygous for driver and Ub 6 .

Article Snippet: Beads were rinsed 10X with RIPA + PI and 3X with kinase buffer, split equally and one side was supplemented with additional PI, whereas the other was supplemented with 100 nM (final) USP2 catalytic domain (Boston Biochem).

Techniques: Construct, Recombinant, Incubation, Western Blot, Molecular Weight, Expressing, Membrane, Modification, Isolation, Ubiquitin Proteomics, Stripping Membranes, Two Tailed Test, Purification

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

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Cortical PSD-lysates from sham and MCAO-treated mice were assessed for CaMKII and PKC downstream target phosphorylation. Phospho-specific vs total protein bands were quantified. GluN2B: * P = 0.0162 from s; nNOS: * P = 0.0046 from s, # P = 0.0426 from s; one-way ANOVA with Bonferroni test; n = 5 animals/group. b Cortical PSD-lysates from sham and MCAO-treated mice were assessed for CaMKII activity (* P < 0.0001, two-tailed unpaired t -test; n = 9 animals/group). c Same as in b , but for PKC (* P < 0.0001, two-tailed unpaired t -test; n = 8 animals/group). d Post-ischemic PSD lysates were treated with recombinant deubiquitinase USP2, and CaMKII activity was reassessed and quantified (* P = 0.0026, two-tailed paired t -test; n = 6 animals). e Same as in d , but for PKC (* P = 0.0012, two-tailed paired t -test; n = 8 animals). c contralateral, i ipsilateral, S serine, s sham. Data are expressed as mean ± s.e.m.

Article Snippet: To determine the role of ubiquitin in regulating kinase activities, lysates and precipitates were treated with recombinant deubiquitinase USP2 catalytic domain (Enzo Life Sciences) for 1 h at 37 °C at a ratio of 1: 5 µg USP2: lysate.

Techniques: Activity Assay, Two Tailed Test, Recombinant

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation levels of Cdk5 downstream targets were determined in PSD lysates of sham and MCAO-treated animals. Tau: * P = 0.0137 from s, # P = 0.0055 from s; Crmp2: * P = 0.0496 from s and P = 0.0430 from c; one-way ANOVA with Bonferroni test; n = 4–5 animals/group. b Cortical PSD-lysates from sham and MCAO-treated mice were assessed for Cdk5 activity (* P < 0.0001, two-tailed unpaired t -test; n = 6 mice/group). c Cdk5 activity was measured in post-ischemic PSD lysates untreated and treated with recombinant deubiquitinase USP2 (* P = 0.0057, two-tailed paired t -test; n = 6 animals). c contralateral, i ipsilateral, S serine, s sham. Data are expressed as mean ± s.e.m.

Article Snippet: To determine the role of ubiquitin in regulating kinase activities, lysates and precipitates were treated with recombinant deubiquitinase USP2 catalytic domain (Enzo Life Sciences) for 1 h at 37 °C at a ratio of 1: 5 µg USP2: lysate.

Techniques: Activity Assay, Two Tailed Test, Recombinant

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation of Pyk2 kinase target proteins was analyzed in PSD extracts. GluN2B: * P = 0.0016 from s and * P = 0.0001 from c; Src: * P < 0.0001 from s and * P = 0.0003 from c; Tau: * P < 0.0001 from s and c; one-way ANOVA with Bonferroni test; n = 5 animals/group. b Pyk2 tyrosine phosphorylation was assessed on Y402 (* P = 0.0184 from s; ** P = 0.0040 from s and ** P < 0.0001 from 30 min rep; one-way ANOVA with Bonferroni test; n = 4–6 animals/group). c Overall Pyk2 tyrosine phosphorylation was assessed after Pyk2 pull down with a phospho-tyrosine-specific antibody (* P = 0.0089 from s; ** P = 0.0005 from s; one-way ANOVA with Bonferroni test; n = 4-6 animals/group). d Phosphorylation of immobilized recombinant his-GST-Src by Pyk2 derived from sham and MCAO lysates untreated or treated with recombinant USP2 was determined. * P = 0.0152 from s; # P = 0.0242 from i/untreated; one-way ANOVA with Bonferroni test; n = 4 animals/group. c contralateral, i ipsilateral, S serine, s sham, Y tyrosine. Data are expressed as mean ± s.e.m.

Article Snippet: To determine the role of ubiquitin in regulating kinase activities, lysates and precipitates were treated with recombinant deubiquitinase USP2 catalytic domain (Enzo Life Sciences) for 1 h at 37 °C at a ratio of 1: 5 µg USP2: lysate.

Techniques: Recombinant, Derivative Assay

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