Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Outline of this study. Comparative analysis of USP7 inhibition or degradation in PDAC and melanoma will be carried out using customized inhibitors and degraders. b. Chemical structures of previously described USP7 inhibitors as well as of NK192 (chimera derived from potent USP7 inhibitors FT671 and Compound 5) which was used as USP7 ligand for the PROTAC library synthesis. Half-maximal inhibition (IC 50 ) values were derived from data shown in panel c. NK264 represents a Biotin-functionalized variant of NK192, which was used for pulldown experiments shown in panel d. c. Ubiquitin-Rhodamine110Gly (Ub-RhoG) cleavage assay to determine inhibitory potency of compounds shown in b. Full length human USP7 was incubated with the respective compounds, the fluorogenic substrate was added and residual activity was read out through fluorescence measurements. d. Competitive pulldown experiment to assess proteome-wide specificity of NK192. Biotin-functionalized NK264 was immobilized on beads. Proteins enriched from Panc89 lysate either treated with DMSO or with NK192 (10 µM, 500x IC 50 ) were quantified by mass spectrometry.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Inhibition, Derivative Assay, Variant Assay, Ubiquitin Proteomics, Cleavage Assay, Incubation, Activity Assay, Fluorescence, Mass Spectrometry

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic of the synthesis of USP7-targeting degraders. Functionalized USP7 ligands (top left, pink triangle) were either coupled directly to VHL-linker conjugates (yellow triangle with grey stick, through steps A and B) or first further functionalized with linkers followed by coupling to the VHL ligand (yellow triangle, through steps A, C and D). See the Supporting Information for detailed chemical procedures. b. Chemical structures of USP7-targeting degrader library. A general structure of the PROTACs is shown on the left, consisting of a USP7 binding ligand (top) and E3 ligase binding ligand (bottom) separated by a linker. Linker attachment points to both ligands are indicated by waved lines (black: USP7, red: VHL). Linkers are shown in the table on the right. c.-d. Assessment of USP7 levels upon treatment with compounds in Panc89 cells (c) and Ma-Mel-47 cells (d). Cells were treated with 5 µM of indicated compounds for 24 h, and cell lysates were analyzed through Western blots with indicated antibodies. See the Supporting Information for uncropped blots. e. Chemical structure of NK225.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Western Blot

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Chemical structures of improved VHL-targeting PROTACs NK250, NK266 and non-VHL binding control compound NK245. The additional methyl groups in the VHL ligand in NK250 and NK266 (leading to enhanced VHL binding) as well as the inversed hydroxyproline stereocenter in NK245 (abrogating VHL binding) are highlighted. b. USP7 degradation assay. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with 5 µM of indicated compounds for 24 h and analyzed by Western blot. See the Supporting Information for uncropped blots. c. Assessment of USP7 degradation efficiency. Panc89 cells (left) or Ma-Mel-47 cells (right) were treated with increasing concentrations of either NK250 (left) or NK266 (right). d. Determination of degradation kinetics. Panc89 cells (left) or Ma-Mel47 cells (right) were treated with of 1 µM of NK250 (left) and NK266 (right) for indicated times. e. Degradation rescue experiment. Panc89 cells (left) or Ma-Mel47 cells (right) were pretreated for 2 h with either DMSO, NEDDylation inhibitor MLN4924 (500 nM), proteasome inhibitor Carfilzomib (CFZ, 250 nM) or VHL ligand NK249 (10 µM) followed by 1 µM of NK250 or N266 for 20 h. f. Chemical structure of VHL ligand NK249. g. Orthogonal confirmation of USP7 depletion by immune fluorescence. Panc89 cells were treated with 1 µM NK250 for 24 h before staining for USP7 (green), actin (red), and with DAPI (blue). Scale bar: 10 µm.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Binding Assay, Control, Degradation Assay, Western Blot, Fluorescence, Staining

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: a. Schematic representation of HiBiT endpoint degradation assay. b. HiBiT-based quantification of USP7 levels. MV4-11 cells stably expressing HiBiT-USP7 generated through lentiviral transduction were treated with PROTACs for either 6 or 24 h. Remaining HiBiT-USP7 could be detected through the luciferase signal using HiBiT Lytic Detection System. Data are shown as mean ± S.D. (N=3), normalized to DMSO treatment. Half-maximal degradation concentrations (DC 50 ) derived from these data are given. c. Schematic representation of a cellular ternary complex formation assay. Halotag-VHL- and NanoLuc-USP7-expressing cells are sequentially treated with Halotag-Fluorophor and PROTAC. Compound-induced proximity is read out by a bioluminescence resonance energy transfer (BRET) signal as shown, demonstrating ternary complex formation. d. Cellular ternary complex formation assay. HEK293T cells overexpressing Halotag-VHL and NanoLuc-USP7 were treated with HaloTag NanoBRET 618 Ligand for 20 h, followed by NK250 or NK266 treatment at indicated concentrations for 2 h prior BRET measurement. The determined half-maximal ternary complex formation concentration (EC 50 ) for NK250 is given. Data are shown as mean ± S.D. (N=4). mBu, milli BRET units.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Degradation Assay, Stable Transfection, Expressing, Generated, Transduction, Luciferase, Derivative Assay, Tube Formation Assay, Bioluminescence Resonance Energy Transfer, Concentration Assay

Journal: bioRxiv

Article Title: Targeted degradation of USP7 in solid cancer cells reveals disparate effects of deubiquitinase inhibition vs. acute protein depletion

doi: 10.1101/2025.07.01.662351

Figure Lengend Snippet: Proteomic analysis of Panc89 cells treated with PROTAC NK250 (a-c) or inhibitor NK192 (d-f) for indicated time points. Volcano blots report proteins identified by data-independent acquisition mass spectrometry in Panc89 cells treated with NK192 (5 µM) or NK250 (1 µM) for 6, 24 or 72 h. Proteins annotated as members of the non-canonical repressive complex 1.6 are highlighted in orange, chromatin bound E3 ligases are highlighted in black, the most upregulated proteins by USP7 inhibition in blue. g.-l. Proteomic analysis of Ma-Mel-47 cells treated with PROTAC NK266 (g-I, 1 µM) or inhibitor NK192 (j-l, 5 µM) for indicated time points.

Article Snippet: HiBiT-USP7 construct was cloned by extraction of USP7 fragment from plasmid pQHA-USP7 WT puroR (Addgene #46753) using appropriate restriction sites.

Techniques: Data-independent acquisition, Mass Spectrometry, Inhibition

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: a His-tag pulldown-HPLC-MS/MS showing that His-TOP1 interacted USP7 under unperturbed condition. After transfection of 6×His-tagged TOP1 expression plasmid or empty vector control (pTrex), HCT116 cells were subjected to His-tag pulldown using Ni-NTA agarose, followed by HPLC-MS. b In vitro assay showing that USP7 reversed TOP1 ubiquitylation. Recombinant TOP1 was subjected to ubiquitylation with ubiquitin, Ube1 (E1), Ubc5Hα (E2), and RNF4 (E3) for 30 min, followed by termination with EDTA and incubation with increasing concentrations of recombinant USP7 for another 30 min. Samples were Western blotted with α-Ub antibody. c His-tag pulldown assay showing that PARGi enhanced TOP1-USP7 interaction. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. His-tag pulldown was performed with Ni-NTA agarose under native conditions. Western blotting was performed with the indicated antibodies. d PLA assay showing TOP1-USP7 interaction in PARGi-treated cells. Following transfection of 6×His-tagged TOP1 expression plasmid and FLAG-USP7 expression plasmid, HEK293 cells were treated as indicated. PLA assays were performed using rabbit α-His-tag antibody and mouse α-FLAG tag antibody. The scale bar represents 10 μm. e Inhibiting USP7 restored TOP1-DPC ubiquitylation in the presence but not in the absence of PARGi. Upper panel : HEK293 cells were treated as indicated: CPT (20 µM, 1 h), CPT + FLAG-USP7 transfection, CPT + USP7i (10 µM, 1 h pre-treatment), CPT + PARGi (10 µM, 1 h pre-treatment), CPT + PARGi + FLAG-USP7 transfection, CPT + PARGi + USP7i. Following treatments, cells were subjected to the modified RADAR assay for detection of TOP1-DPCs and their ubiquitylation using α-TOP1 and α-Ub antibodies. Lower panel : densitometric quantitation of ubiquitylated TOP1-DPC signals generated from triplicate experiments including representative blots shown in ( c ) using ImageJ. n = 3 independent experiments. Data are presented as mean values +/− standard deviation (SD). P value was calculated by paired Student’s t-test (two-tailed distribution). *: p = . NS not significant. f Inhibiting USP7 did not impact the induction of γH2AX upon exposure to CPT. U2OS cells were synchronized in the S phase by double thymidine and treated with CPT (1 µM) in the absence of presence of USP7i (10 µM, 1 h pre-treatment) and collected for IF by iSIM using an anti-γH2AX antibody. The scale bar represents 10 μm.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Tandem Mass Spectroscopy, Transfection, Expressing, Plasmid Preparation, Control, In Vitro, Recombinant, Ubiquitin Proteomics, Incubation, Western Blot, FLAG-tag, Modification, Quantitation Assay, Generated, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

doi: 10.1038/s41467-021-25252-9

Figure Lengend Snippet: a PARP1 and TOP1 form cellular protein complexes (present study and refs. , ). b TOP1-DPC trapped by CPT is rapidly modified with PAR by PARP1 and with ubiquitin (by RNF4 and potentially other E3 ligases, not shown). The PARylation recruits TDP1, PARG, and USP7 to the TOP1-DPC. c TOP1-DPC PARylation is readily and rapidly reversed by PARG, enabling the 26S proteasome to target the ubiquitylated TOP1-DPC for degradation. d TDP1 hydrolyzes the TOP1 peptide to expose the DNA ends for repair. e In the presence of PARGi, TOP1-DPC dePARylation is blocked and the persistent PAR polymers on TOP1-DPC obstruct the proteasome hence stabilize TOP1-DPC. f The stabilization of TOP1-DPC triggers USP7 to deubiquitylate the DPC to recycle the ubiquitin molecules.

Article Snippet: Human recombinant USP7 was purchased from R&D Systems (cat # E-519).

Techniques: Modification, Ubiquitin Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Therapeutic inhibition of USP7 promotes antitumor immune responses

doi: 10.1136/jitc-2025-012287

Figure Lengend Snippet: Characteristics of a novel and potent ubiquitin-specific protease 7 inhibitor. ( A ) Chemical structure of OAT-4828. ( B ) OAT-4828 activity determined in the enzymatic assay with a fluorescently labeled ubiquitin as a substrate (rhodamine 110), graph presents means from two independent repetitions (n=2). ( C ) OAT-4828 activity determined in the reporter assay (Ub-CHOP reporter system) with the ubiquitinated protein as a substrate, graph presents means from two independent repetitions (n=2). ( D ) The selectivity of OAT-4828 determined in the enzymatic test engaging 15N ubiquitin and 10 µM concentration of OAT-4828, (n=2). ( E ) Inhibition of β-catenin-dependent Wnt signaling pathway in SW480 cell line, measured using TOPFlash and FOPFlash Wnt/β-catenin activity assays after 24 hours incubation with OAT-4828, the experiment was repeated three times and the representative graph is shown. ( F ) Graph illustrates the plasma concentration-time profile of OAT-4828 following a single oral dose administration at 50 mg/kg and 100 mg/kg in mice. Both total and free (unbound) drug concentrations are depicted, n=2. IC50 defined as the concentration that reduces enzyme activity by 50 percent, EC50 defined as the concentration that reduces enzyme activity by 50 percent, PO, oral administration.

Article Snippet: The assay included: USP7 enzyme (0.4 nM final concentration, R&D Systems, Cat# E-519), OATD-4828 compound (from serial dilution), and Ub-Rho110Gly substrate (500 nM final concentration, UbiQ Bio, Cat# UbiQ-002).

Techniques: Ubiquitin Proteomics, Activity Assay, Enzymatic Assay, Labeling, Reporter Assay, Concentration Assay, Inhibition, Incubation, Clinical Proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 1. An RNAi screen for suppressors of aggregation of mutated SOD1G85R-YFP in C. elegans identifies math-33 as a strong suppressor of aggregation. (A) C. elegans MATH-33 and its homologs in Drosophila and human, Usp7 and USP7, respectively, share all major protein domains. These include the Meprin And Traf Homology domain (MATH), catalytic peptidase domain, herpes simplex virus ICP0 protein binding domain (ICP0), and C2 terminal domain. The position of a deletion mutation, ok2974, in the C. elegans mutant is indicated with a red bar. (B) The math-33(ok2974) mutant worms showed reduced aggregation of SOD1G85R-YFP, as evidenced by the reduced fluorescent signal in head neurons when compared to the controls on the WT background. (Scale bar, 5 μm.) (C) Western blot analysis of soluble (S) and pellet (P) protein fractions from the WT and math-33(ok2974) mutant worms. Representative immunoblots of SOD1G85R-YFP (Left) and quantification of SOD1G85R-YFP protein from the S (Middle; *P < 0.05) and P (Right; *P < 0.05) fractions are shown (n = 3). (D) qRT- PCR analysis of the mRNA expression levels of the SOD1G85R transgene in math-33(ok2974) mutant C. elegans as compared to the control background (WT) (n = 3; n.s., nonsignificant). (E) Locomotor behavior, as measured by the thrashing assay, of C. elegans with neuronal expression of SOD1G85R, with or without the suppressor mutation math-33(ok2974), as compared to control C. elegans expressing SOD1WT (n = 9; ***P < 0.001, ****P < 0.0001). Error bars indicate ± SEM.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Virus, Protein Binding, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 2. A reduction in the USP7 function increases clearance of misfolded proteins. (A) Western blot analysis of cell lysates derived from mock (CTRL) or USP7 knockdown in HEK293 cells expressing SOD1G85R. USP7 knockdown reduced the SOD1G85R proteins in both the supernatant (S) and pellet (P) fractions, without affecting the level of endogenous WT SOD1 (SOD1WT). Quantification of SOD1WT and SOD1G85R protein levels by immunoblotting is shown (n = 3; ***P < 0.001, ****P < 0.0001; n.s., nonsignificant). (B) A small-molecule inhibitor of USP7, HBX41108, reduced the level of misfolded SOD1G85R protein, but not that of SOD1WT protein in HEK293 cells. A dose-dependent effect on the level of SOD1G85R protein with increasing concentrations of HBX41108, as compared to the vehicle control (DMSO), was observed in the supernatants of the cell lysates (n = 3; n.s., nonsignificant; *P < 0.05). (C) Western blots of a representative cycloheximide chase experiment to determine the SOD1 protein half-life in the USP7 knockdown cells versus controls (Left). Quantification of the SOD1G85R clearance as analyzed by immunoblotting is shown (Right). The graph indicates the relative band intensity of SOD1G85R at each chase time point in the USP7 knockdown cells versus controls (n = 3; *overall P < 0.05 for the three time points after 0 h). Error bars indicate ± SEM.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Western Blot, Derivative Assay, Knockdown, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 3. USP7 regulates misfolded mutant SOD1 protein levels through the transcription factor SMAD2. (A) The SMAD transcriptional activity was increased in HEK293 cells upon USP7 knockdown (Left) or inhibition of USP7 by the small-molecule drug HBX41108 at 2 μM (Right), as measured by the SMAD response element (SMAD-RE)–mediated luciferase activity assay (n = 3; *P < 0.05, ****P < 0.0001). (B) The protein levels of SMAD2, but not those of SMAD3, were increased upon USP7 knockdown (n = 3; **P < 0.01). (C) Endogenous SMAD2 was coimmunoprecipitated when Flag-USP7 expressed in HEK293 cell was pulled down by the anti-Flag antibody but not the IgG control. (D) Immunoblot analysis of SOD1G85R in cells with USP7 or SMAD2 knockdown, or double knockdown, indicated that loss of SMAD2 abolished the effect of USP7 on the regulation of SOD1G85R levels in both the supernatant and pellet fractions (n = 4; *P < 0.05, **P < 0.01). Error bars indicate ± SEM.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Mutagenesis, Activity Assay, Knockdown, Inhibition, Luciferase, Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 4. USP7 deubiquitinates NEDD4L, the E3 ligase for SMAD2. (A) Immunoblots and quantification of endogenous NEDD4L in HEK293 cells with USP7 or control knockdown (n = 3; *P < 0.05). (B) Endogenous NEDD4L was coimmunoprecipitated when Flag-USP7 expressed in HEK293 cells was pulled down by the anti-Flag antibody but not the IgG control. (C) In the in vitro deubiquitination assay, V5-NEDD4L expressed in HEK293 cells was immunoprecipitated and used as a substrate for incubation with purified GST-USP7 or GST. The presence of USP7 significantly decreased the levels of ubiquitinated NEDD4L protein (n = 3; *P < 0.05). Error bars indicate ± SEM. (D) In the in vivo deubiquitination assay, cell lysates expressing Flag-USP7 variants, V5-NEDD4L, and HA-Ub and were incubated with anti-V5 antibody to pull down NEDD4L, and the immunoprecipitates were subjected to analysis of ubiquitination by immunoblotting. C223S is a catalytic mutant of USP7, and GUS is a control.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Western Blot, Control, Knockdown, In Vitro, Immunoprecipitation, Incubation, Purification, In Vivo, Expressing, Ubiquitin Proteomics, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 6. The knockdown of Drosophila Usp7 suppresses neurotoxicity in- duced by mutant SOD1 or TDP-43. (A) Chart of the climbing (negative geo- taxis) assay in adult Drosophila expressing human SOD1A4V in motor neurons (Left). Quantification of the climbing ability of Drosophila expressing human SOD1A4V in motor neurons, together with gene-specific RNAi against Usp7 (34708), Smox (26756), Babo (40866), or control RNAi (Right; n = 8 indepen- dent groups; **P < 0.01). (B) Reduction in Usp7 by RNAi (34708) strongly suppresses the eye degeneration phenotypes in TDP-43M337V strains, while reduction in Smox (RNAi 26756) or Babo (RNAi 40866) worsens the eye de- generation phenotypes, when compared with the control Luc RNAi strain (Left). (Scale bar, 100 μm.) Quantification of pigment content in adult eyes confirms the protection against degeneration by a loss of Usp7 in TDP-43M337V

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Knockdown, Mutagenesis, Expressing, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 5. USP7 regulates autophagy through SMAD2. (A) Immunoblots of LC3II proteins in HEK293 cells after mock or SMAD2 knockdown, with or without 200 nM bafilomycin A1 treatment. Quantification of LC3II is shown (n = 6; *P < 0.05). (B) Immunoblots and quantification of LC3II protein levels after mock or USP7 knockdown, with or without bafilomycin A1 treatment (n = 8; *P < 0.05). (C) Immunoblots and quantification of LC3II protein levels after treatment with the USP7 inhibitor HBX41108, with or without bafilomycin A1 (n = 6; *P < 0.05). (D) Immunoblots and quantification of LC3II protein levels after single or double knockdown of USP7 and SMAD2, with or without bafilomycin A1 (n = 6; *P < 0.05). Error bars indicate ± SEM.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Western Blot, Knockdown

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

doi: 10.1073/pnas.2014349117

Figure Lengend Snippet: Fig. 7. Role of the USP7–SMAD2 pathway in neurons and patient tissues. (A) Mouse neurons were transduced with SOD1G85R or SOD1WT HSVs and treated with vehicle (DMSO) or the USP7 inhibitor HBX41108. (Scale bar, 150 μm.) Live cells were loaded with the calcein AM dye to determine cell viability (Left). Quantification of the cell viability (Right; n = 8; n.s., nonsig- nificant; *P < 0.05). (B) Representative Western blotting of human spinal cord tissue lysates derived from ALS patients and controls show up- regulation of SMAD2 in the patients’ tissues (Left). Quantification of the SMAD2 protein levels (Right; n = 8 for control and n = 24 for ALS; **P < 0.01). Error bars indicate ± SEM.

Article Snippet: The plasmids expressing USP7 shRNAs with a red fluorescent protein (RFP) marker (pRFP-C-RS, catalog no. TF308454) and the scrambled control (catalog no. TR30015) were from OriGene Technologies Inc.

Techniques: Transduction, Western Blot, Derivative Assay, Control