Review



ubiquitin conjugating enzyme  (MedChemExpress)


Bioz Verified Symbol MedChemExpress is a verified supplier
Bioz Manufacturer Symbol MedChemExpress manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 93
      Buy from Supplier

    Structured Review

    MedChemExpress ubiquitin conjugating enzyme
    Ubiquitin Conjugating Enzyme, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/UbcH7%2FUBE2L3%2C+Human/pm41296569-389-57-61
    Average 93 stars, based on 1 article reviews
    ubiquitin conjugating enzyme - by Bioz Stars, 2026-10
    93/100 stars

    Images

    Related Articles

    In Vitro:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Ubiquitin Proteomics:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Expressing:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Clinical Proteomics:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Immunoprecipitation:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Modification:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Incubation:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Conjugation Assay:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.

    Western Blot:

    Article Title: Glutamine synthetase modulates YAP activation by stabilizing LATS under glutamine homeostasis.
    Article Snippet: Subsequently, the cells were incubated with Alexa-Fluor 488-conjugated anti-mouse (Thermo, A-10680; 1:1000) secondary antibody for 1 h at room temperature. .. For in vitro ubiquitination, 1 mg of protein lysate extracted from cells expressing CSF-LATS1 was immunoprecipitated by S-beads, washed 4 times with modified NETN buffer, followed by incubation in 30 μL of ubiquitination conjugation reaction buffer [2 mM ATP (Med Chem Express), 0.5 mg His-Ubiquitin (Med Chem Express), 0.1 mM ubiquitin-activating enzyme (E1, UBE1,YEASEN), and 0.5 mM ubiquitin-conjugating enzyme (E2, UbcH7, Med Chem Express) in the absence or presence of MBP-WWP1 (E3) or HAGS at 30◦C for 90 min. Then, the mixture was washed 4 times with NETN buffer and subjected to 1×SDS-PAGE for immunoblotting. .. Immunofluorescence images were captured using an Olympus confocal microscope.



    Similar Products

    ubiquitin conjugating enzyme  (MedChemExpress)
    93
    MedChemExpress ubiquitin conjugating enzyme
    Ubiquitin Conjugating Enzyme, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/UbcH7%2FUBE2L3%2C+Human/pm41296569-389-57-61
    Average 93 stars, based on 1 article reviews
    ubiquitin conjugating enzyme - by Bioz Stars, 2026-10
    93/100 stars
    		  Buy from Supplier
    ubiquitin conjugating enzyme e2 c ube2c  (Proteintech)
    93
    Proteintech ubiquitin conjugating enzyme e2 c ube2c
    Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, <t>UBE2C,</t> and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; <t>UBE2C,</t> ubiquitin-conjugating enzyme E2 C.
    Ubiquitin Conjugating Enzyme E2 C Ube2c, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/UBE2C+Antibody/pmc12964017-106-10-19
    Average 93 stars, based on 1 article reviews
    ubiquitin conjugating enzyme e2 c ube2c - by Bioz Stars, 2026-10
    93/100 stars
    		  Buy from Supplier
    ubiquitin conjugating enzyme e2  (Unigene)
    86
    Unigene ubiquitin conjugating enzyme e2
    Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, <t>UBE2C,</t> and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; <t>UBE2C,</t> ubiquitin-conjugating enzyme E2 C.
    Ubiquitin Conjugating Enzyme E2, supplied by Unigene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/conjugating+e2+enzyme+ubiquitin/pm41382314-318-41-44
    Average 86 stars, based on 1 article reviews
    ubiquitin conjugating enzyme e2 - by Bioz Stars, 2026-10
    86/100 stars
    		  Buy from Supplier
    ubiquitin-conjugating enzyme h5c ubch5c  (Boston Biochem)
    90
    Boston Biochem ubiquitin-conjugating enzyme h5c ubch5c
    Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, <t>UBE2C,</t> and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; <t>UBE2C,</t> ubiquitin-conjugating enzyme E2 C.
    Ubiquitin Conjugating Enzyme H5c Ubch5c, supplied by Boston Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/ubch5c+e2+627/pm40408472-312-29-34
    Average 90 stars, based on 1 article reviews
    ubiquitin-conjugating enzyme h5c ubch5c - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    ubiquitin-conjugating enzyme ube2n  (Proteostasis Therapeutics)
    90
    Proteostasis Therapeutics ubiquitin-conjugating enzyme ube2n
    Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, <t>UBE2C,</t> and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; <t>UBE2C,</t> ubiquitin-conjugating enzyme E2 C.
    Ubiquitin Conjugating Enzyme Ube2n, supplied by Proteostasis Therapeutics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/ubiquitin+conjugating+enzyme+ube2n/10__1172_slash_jci184665-10-0-4
    Average 90 stars, based on 1 article reviews
    ubiquitin-conjugating enzyme ube2n - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    human e2 ubiquitin conjugating enzymes  (R&D Systems)
    96
    R&D Systems human e2 ubiquitin conjugating enzymes
    A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 <t>ubiquitin</t> activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to <t>E2</t> to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.
    Human E2 Ubiquitin Conjugating Enzymes, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/Recombinant+Human+Ubiquitin+Protein%2C+CF/pmc12007708-308-25-42
    Average 96 stars, based on 1 article reviews
    human e2 ubiquitin conjugating enzymes - by Bioz Stars, 2026-10
    96/100 stars
    		  Buy from Supplier
    ubiquitin-conjugating enzyme (ube2)  (Yeasen Biotechnology)
    90
    Yeasen Biotechnology ubiquitin-conjugating enzyme (ube2)
    A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 <t>ubiquitin</t> activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to <t>E2</t> to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.
    Ubiquitin Conjugating Enzyme (Ube2), supplied by Yeasen Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/ubiquitin+conjugating+enzyme++ube2+/pmc11980547-303-24-27
    Average 90 stars, based on 1 article reviews
    ubiquitin-conjugating enzyme (ube2) - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier
    ube2d ubiquitin-conjugating enzymes  (Proteostasis Therapeutics)
    90
    Proteostasis Therapeutics ube2d ubiquitin-conjugating enzymes
    ( A ) GFP-tagged huntingtin-polyQ (Htt-polyQ72-GFP) driven with GMR-Gal4 leads to GFP-fluorescent Htt protein aggregates in the retina at 30 days of age. Compared to negative controls (mcherry RNAi and luciferase RNAi ), RNAi for E2 ubiquitin-conjugating enzymes, associated deubiquitinating enzymes (DUBs), and E3 ubiquitin ligases (E3s) modulates the amount of Htt-polyQ72-GFP aggregates. Specifically, RNAi for Ubc6 (homologous of UBE2A/B) and for its associated E3 ubiquitin ligases Ubr4 and Kcmf1 increases the amount of Htt-polyQ72-GFP protein aggregates. Similar increases are also seen with the knockdown of <t>UBE2D/eff</t> and the associated E3 HUWE1, and with RNAi for Ubc10/UBE2L3, Ubc84D/UBE2L6, and for the associated E3 enzyme BTBD8. Positive controls include Akt RNAi, which reduces protein aggregates, and Hsp83 RNAi, which increases them. ( B , C ) Quantitation of the total area of Htt-polyQ72-GFP aggregates modulated by RNAi for E2s (B) and for associated E3s and DUBs (C). Relative fold changes compared to control RNAi interventions are shown; n = 5 (biological replicates), SD. Each data point in the graph represents a single RNAi targeting the corresponding gene. For each RNAi, the mean of 5 biological replicates is shown, with each biological replicate representing a single eye (each from a distinct animal). The variability in the analysis of each single gene is likely due to the distinct efficacy of the different RNAi lines targeting the same gene. ( D ) qRT-PCR indicates that there are no changes in GFP and Htt mRNA levels upon eff knockdown compared to control RNAi, indicating that eff RNAi does not modulate the amount of GFP-tagged huntingtin-polyQ aggregates via changes in the expression of Htt-polyQ72-GFP transgenes; n = 3 (biological replicates) and SEM. ( E ) Levels of Htt-polyQ72-GFP aggregates detected by western blot with anti-GFP antibodies identify Htt-polyQ72-GFP monomers (~50 kDa) and HMW assemblies of Htt-polyQ72-GFP in the stacking gel (>250 kDa). Knockdown of UBE2D/eff increases the levels of HMW Htt-polyQ72-GFP; n = 3 (biological replicates), SEM, and p -values (one-way ANOVA) are indicated, with ** p < 0.01, compared to mcherry RNAi . See also . The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in Images.
    Ube2d Ubiquitin Conjugating Enzymes, supplied by Proteostasis Therapeutics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubiquitin+conjugating+enzyme/ube2d+eff/pmc11778781-33-23-2
    Average 90 stars, based on 1 article reviews
    ube2d ubiquitin-conjugating enzymes - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier

    Image Search Results


    Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, UBE2C, and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; UBE2C, ubiquitin-conjugating enzyme E2 C.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Multiomics Profiling Reveals Distinct Immunosuppression and Metabolic Dysregulation in Aggressive Subtypes of Thyroid Cancer

    doi: 10.1016/j.mcpro.2026.101513

    Figure Lengend Snippet: Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, UBE2C, and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; UBE2C, ubiquitin-conjugating enzyme E2 C.

    Article Snippet: Primary antibodies—including FCGR2A (RRID: AB_2246912 , Cat.15625-1-AP, Proteintech, 1:600 dilution), ubiquitin-conjugating enzyme E2 C (UBE2C) (RRID: AB_11232220 , Cat.66087-1-Ig, Proteintech, 1:500), and nucleotide-binding protein-like (NUBPL) (RRID: AB_2878402 , Cat.17393-1-AP, Proteintech, 1:200)—were diluted in antibody dilution buffer and applied to tissue sections.

    Techniques: Single Cell, RNA Sequencing, Expressing, Biomarker Discovery, Immunohistochemistry, Binding Assay, Ubiquitin Proteomics

    A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 ubiquitin activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to E2 to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 ubiquitin activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to E2 to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Ubiquitin Proteomics, Activity Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Negative Control, Quantitative Proteomics

    A) Protein expression and purification. Coomassie stained SDS-PAGE gel of purified proteins: E1 activating enzyme (PF3D7_1225800), E2 conjugating enzymes (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, and PF3D7_1356300). B) In vitro autoubiquitination by P.falciparum E2 enzymes. Western blot of ubiquitination reaction catalysed by select PfE2 enzymes, visualised by anti-HA antibody. PfE2 enzymes were incubated with ATP, PfE1, and HA-Ub. The products of the reaction were resolved and visualised to reveal signature ubiquitination patterns formed by the unique activity of the different E2 enzymes.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: A) Protein expression and purification. Coomassie stained SDS-PAGE gel of purified proteins: E1 activating enzyme (PF3D7_1225800), E2 conjugating enzymes (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, and PF3D7_1356300). B) In vitro autoubiquitination by P.falciparum E2 enzymes. Western blot of ubiquitination reaction catalysed by select PfE2 enzymes, visualised by anti-HA antibody. PfE2 enzymes were incubated with ATP, PfE1, and HA-Ub. The products of the reaction were resolved and visualised to reveal signature ubiquitination patterns formed by the unique activity of the different E2 enzymes.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Expressing, Purification, Staining, SDS Page, In Vitro, Western Blot, Ubiquitin Proteomics, Incubation, Activity Assay

    A) Comparison of PfHEUL to orthologs in model organisms. Cartoon alignment of PfHEUL with human (Q7Z6Z7), S.cerevisiae (Q03280) and A.thaliana (Q8GY23) orthologs showing conservation of HECT and DUF908 domains. B) In vitro autoubiquitination of PfHEUL HECT domain using human E2 enzymes. Anti-HA western blot representing the in vitro autoubiquitination assay used to validate the activity of the PfHEUL HECT domain using human HA-tagged ubiquitin, E1 activating (UBA1), and E2 conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3). All E2 enzymes except UBE2E1 were capable of functioning in concert with PfHEUL HECT. C) In vitro autoubiquitination of PfHEUL HECT domain using P.falciparum E2 enzymes. Western blots of the in vitro autoubiquitination assay using P. falciparum ubiquitination components: E1 activating enzyme (PF3D7_1225800), indicated E2 conjugating enzymes, and the PfHEUL HECT domain. (Left) Western blot using anti-ubiquitin antibodies to identify ubiquitin conjugated proteins following the ubiquitination reaction. (Right) Western blot using mouse derived PfHEUL HECT antibodies to specifically identify PfHEUL HECT in its native and ubiquitinated states.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: A) Comparison of PfHEUL to orthologs in model organisms. Cartoon alignment of PfHEUL with human (Q7Z6Z7), S.cerevisiae (Q03280) and A.thaliana (Q8GY23) orthologs showing conservation of HECT and DUF908 domains. B) In vitro autoubiquitination of PfHEUL HECT domain using human E2 enzymes. Anti-HA western blot representing the in vitro autoubiquitination assay used to validate the activity of the PfHEUL HECT domain using human HA-tagged ubiquitin, E1 activating (UBA1), and E2 conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3). All E2 enzymes except UBE2E1 were capable of functioning in concert with PfHEUL HECT. C) In vitro autoubiquitination of PfHEUL HECT domain using P.falciparum E2 enzymes. Western blots of the in vitro autoubiquitination assay using P. falciparum ubiquitination components: E1 activating enzyme (PF3D7_1225800), indicated E2 conjugating enzymes, and the PfHEUL HECT domain. (Left) Western blot using anti-ubiquitin antibodies to identify ubiquitin conjugated proteins following the ubiquitination reaction. (Right) Western blot using mouse derived PfHEUL HECT antibodies to specifically identify PfHEUL HECT in its native and ubiquitinated states.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Comparison, In Vitro, Western Blot, Activity Assay, Ubiquitin Proteomics, Derivative Assay

    A) Analysis of PfHEUL HECT domain and catalytic cysteine. AlphaFold predicted structure of PfHEUL HECT domain (residues 8211-8591) (green) overlaid with crystal structure human HUWE1 (PDB:7JQ9) (blue) and with catalytic cysteines marked by the red arrow. B) Alignment of PfHEUL and human HUWE1 HECT domains. Rendered in ESPript with catalytic cysteine demarcated by the red arrow. C) Circular Dichroism spectra of wild type (WT) and C8558A mutant PHEUL HECT recombinant protein. D) In vitro ubiquitination assay. Anti-HA western blot of an in vitro ubiquitination assay using wild type and C8558A mutant PfHEUL HECT domains to demonstrate the contribution of C8558 to the transthiolation of ubiquitin, and subsequent ubiquitination of lysine side chains.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: A) Analysis of PfHEUL HECT domain and catalytic cysteine. AlphaFold predicted structure of PfHEUL HECT domain (residues 8211-8591) (green) overlaid with crystal structure human HUWE1 (PDB:7JQ9) (blue) and with catalytic cysteines marked by the red arrow. B) Alignment of PfHEUL and human HUWE1 HECT domains. Rendered in ESPript with catalytic cysteine demarcated by the red arrow. C) Circular Dichroism spectra of wild type (WT) and C8558A mutant PHEUL HECT recombinant protein. D) In vitro ubiquitination assay. Anti-HA western blot of an in vitro ubiquitination assay using wild type and C8558A mutant PfHEUL HECT domains to demonstrate the contribution of C8558 to the transthiolation of ubiquitin, and subsequent ubiquitination of lysine side chains.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Circular Dichroism, Mutagenesis, Recombinant, In Vitro, Ubiquitin Proteomics, Western Blot

    All 13 cysteine residues in Pf3D7_0811400 are labelled and highlighted in green. The C-terminal glycine (Gly76) of ubiquitin interacts with C572, which acts as the catalytic cysteine in the core of the catalytic cleft.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: All 13 cysteine residues in Pf3D7_0811400 are labelled and highlighted in green. The C-terminal glycine (Gly76) of ubiquitin interacts with C572, which acts as the catalytic cysteine in the core of the catalytic cleft.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Ubiquitin Proteomics

    A) Biotin-Ub-Dha-reactivity of wild type and C572R proteins. Wild-type and C572R mutant proteins were expressed in HEK293T cells, purified using anti-FLAG beads and eluted by incubation with a 3XFLAG peptide solution. Purified proteins were subjected to probe labelling in the presence or absence of ATP. (Images of full membranes are included in ). B) Ubiquitin-HA-reactivity of wild type and C572R proteins. Purified FLAG-tagged Pf3D7_0811400 or the C572R mutant was used in an in vitro auto-ubiquitination assay. HA-tagged ubiquitin was added to allow detection by anti-HA immunoblot (left panel). The arrow highlights the weak and transient, but highly specific, formation of a thioester-linked E2 ubiquitin (E2~Ub) conjugate in the wild-type E2. PF3D7_0921000, a Plasmodium E2 characterised earlier in this paper, and UB2R1, a human E2, were used as positive controls (right label). C) Comparison of Pf3D7_0811400 with other P. falciparum E2 enzymes. Sequence alignment showing the conserved catalytic HPN motif in all of the described Pf E2 enzymes but not in Pf3D7_0811400.

    Journal: PLOS Pathogens

    Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

    doi: 10.1371/journal.ppat.1013032

    Figure Lengend Snippet: A) Biotin-Ub-Dha-reactivity of wild type and C572R proteins. Wild-type and C572R mutant proteins were expressed in HEK293T cells, purified using anti-FLAG beads and eluted by incubation with a 3XFLAG peptide solution. Purified proteins were subjected to probe labelling in the presence or absence of ATP. (Images of full membranes are included in ). B) Ubiquitin-HA-reactivity of wild type and C572R proteins. Purified FLAG-tagged Pf3D7_0811400 or the C572R mutant was used in an in vitro auto-ubiquitination assay. HA-tagged ubiquitin was added to allow detection by anti-HA immunoblot (left panel). The arrow highlights the weak and transient, but highly specific, formation of a thioester-linked E2 ubiquitin (E2~Ub) conjugate in the wild-type E2. PF3D7_0921000, a Plasmodium E2 characterised earlier in this paper, and UB2R1, a human E2, were used as positive controls (right label). C) Comparison of Pf3D7_0811400 with other P. falciparum E2 enzymes. Sequence alignment showing the conserved catalytic HPN motif in all of the described Pf E2 enzymes but not in Pf3D7_0811400.

    Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

    Techniques: Mutagenesis, Purification, Incubation, Ubiquitin Proteomics, In Vitro, Western Blot, Comparison, Sequencing

    ( A ) GFP-tagged huntingtin-polyQ (Htt-polyQ72-GFP) driven with GMR-Gal4 leads to GFP-fluorescent Htt protein aggregates in the retina at 30 days of age. Compared to negative controls (mcherry RNAi and luciferase RNAi ), RNAi for E2 ubiquitin-conjugating enzymes, associated deubiquitinating enzymes (DUBs), and E3 ubiquitin ligases (E3s) modulates the amount of Htt-polyQ72-GFP aggregates. Specifically, RNAi for Ubc6 (homologous of UBE2A/B) and for its associated E3 ubiquitin ligases Ubr4 and Kcmf1 increases the amount of Htt-polyQ72-GFP protein aggregates. Similar increases are also seen with the knockdown of UBE2D/eff and the associated E3 HUWE1, and with RNAi for Ubc10/UBE2L3, Ubc84D/UBE2L6, and for the associated E3 enzyme BTBD8. Positive controls include Akt RNAi, which reduces protein aggregates, and Hsp83 RNAi, which increases them. ( B , C ) Quantitation of the total area of Htt-polyQ72-GFP aggregates modulated by RNAi for E2s (B) and for associated E3s and DUBs (C). Relative fold changes compared to control RNAi interventions are shown; n = 5 (biological replicates), SD. Each data point in the graph represents a single RNAi targeting the corresponding gene. For each RNAi, the mean of 5 biological replicates is shown, with each biological replicate representing a single eye (each from a distinct animal). The variability in the analysis of each single gene is likely due to the distinct efficacy of the different RNAi lines targeting the same gene. ( D ) qRT-PCR indicates that there are no changes in GFP and Htt mRNA levels upon eff knockdown compared to control RNAi, indicating that eff RNAi does not modulate the amount of GFP-tagged huntingtin-polyQ aggregates via changes in the expression of Htt-polyQ72-GFP transgenes; n = 3 (biological replicates) and SEM. ( E ) Levels of Htt-polyQ72-GFP aggregates detected by western blot with anti-GFP antibodies identify Htt-polyQ72-GFP monomers (~50 kDa) and HMW assemblies of Htt-polyQ72-GFP in the stacking gel (>250 kDa). Knockdown of UBE2D/eff increases the levels of HMW Htt-polyQ72-GFP; n = 3 (biological replicates), SEM, and p -values (one-way ANOVA) are indicated, with ** p < 0.01, compared to mcherry RNAi . See also . The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in Images.

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: ( A ) GFP-tagged huntingtin-polyQ (Htt-polyQ72-GFP) driven with GMR-Gal4 leads to GFP-fluorescent Htt protein aggregates in the retina at 30 days of age. Compared to negative controls (mcherry RNAi and luciferase RNAi ), RNAi for E2 ubiquitin-conjugating enzymes, associated deubiquitinating enzymes (DUBs), and E3 ubiquitin ligases (E3s) modulates the amount of Htt-polyQ72-GFP aggregates. Specifically, RNAi for Ubc6 (homologous of UBE2A/B) and for its associated E3 ubiquitin ligases Ubr4 and Kcmf1 increases the amount of Htt-polyQ72-GFP protein aggregates. Similar increases are also seen with the knockdown of UBE2D/eff and the associated E3 HUWE1, and with RNAi for Ubc10/UBE2L3, Ubc84D/UBE2L6, and for the associated E3 enzyme BTBD8. Positive controls include Akt RNAi, which reduces protein aggregates, and Hsp83 RNAi, which increases them. ( B , C ) Quantitation of the total area of Htt-polyQ72-GFP aggregates modulated by RNAi for E2s (B) and for associated E3s and DUBs (C). Relative fold changes compared to control RNAi interventions are shown; n = 5 (biological replicates), SD. Each data point in the graph represents a single RNAi targeting the corresponding gene. For each RNAi, the mean of 5 biological replicates is shown, with each biological replicate representing a single eye (each from a distinct animal). The variability in the analysis of each single gene is likely due to the distinct efficacy of the different RNAi lines targeting the same gene. ( D ) qRT-PCR indicates that there are no changes in GFP and Htt mRNA levels upon eff knockdown compared to control RNAi, indicating that eff RNAi does not modulate the amount of GFP-tagged huntingtin-polyQ aggregates via changes in the expression of Htt-polyQ72-GFP transgenes; n = 3 (biological replicates) and SEM. ( E ) Levels of Htt-polyQ72-GFP aggregates detected by western blot with anti-GFP antibodies identify Htt-polyQ72-GFP monomers (~50 kDa) and HMW assemblies of Htt-polyQ72-GFP in the stacking gel (>250 kDa). Knockdown of UBE2D/eff increases the levels of HMW Htt-polyQ72-GFP; n = 3 (biological replicates), SEM, and p -values (one-way ANOVA) are indicated, with ** p < 0.01, compared to mcherry RNAi . See also . The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in Images.

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Luciferase, Ubiquitin Proteomics, Knockdown, Quantitation Assay, Control, Quantitative RT-PCR, Expressing, Western Blot

    (A) TMT mass spectrometry of Drosophila skeletal muscle from old versus young w 1118 flies at 8 vs. 1 week-old; n = 5 biological replicates. The x -axis reports the log 2 FC, whereas the y -axis reports the–log 10 ( p -value). The E1 enzyme UBA1 and the E2 enzymes that are detected are indicated, including UBE2D/eff; n = 5 (biological replicates). See also . (B) The protein levels of UBE2D/eff significantly decline with aging (8 vs. 1 week old) in skeletal muscle; n = 5 (biological replicates), SD, and p -values (Student’s t test). ( C, D ) Immunostaining of Drosophila skeletal muscle at 3 weeks of age indicates that UBE2D/eff RNAi impairs protein quality control, as indicated by the higher age-related accumulation of aggregates of poly-ubiquitinated proteins, compared to control RNAi. The scale bar is 20 μm. In (C), n = 10 (biological replicates), SD, and p -values (Student’s t test). ( E, F ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that eff RNAi impedes proteostasis, as indicated by higher levels of detergent-soluble and insoluble poly-ubiquitinated proteins compared to control RNAi (E). A similar increase is also found for the detergent-soluble levels of p62/Ref(2)P (E). n = 3 (biological replicates), SEM, ** p < 0.01, ns = not significant (one-way ANOVA). The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: (A) TMT mass spectrometry of Drosophila skeletal muscle from old versus young w 1118 flies at 8 vs. 1 week-old; n = 5 biological replicates. The x -axis reports the log 2 FC, whereas the y -axis reports the–log 10 ( p -value). The E1 enzyme UBA1 and the E2 enzymes that are detected are indicated, including UBE2D/eff; n = 5 (biological replicates). See also . (B) The protein levels of UBE2D/eff significantly decline with aging (8 vs. 1 week old) in skeletal muscle; n = 5 (biological replicates), SD, and p -values (Student’s t test). ( C, D ) Immunostaining of Drosophila skeletal muscle at 3 weeks of age indicates that UBE2D/eff RNAi impairs protein quality control, as indicated by the higher age-related accumulation of aggregates of poly-ubiquitinated proteins, compared to control RNAi. The scale bar is 20 μm. In (C), n = 10 (biological replicates), SD, and p -values (Student’s t test). ( E, F ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that eff RNAi impedes proteostasis, as indicated by higher levels of detergent-soluble and insoluble poly-ubiquitinated proteins compared to control RNAi (E). A similar increase is also found for the detergent-soluble levels of p62/Ref(2)P (E). n = 3 (biological replicates), SEM, ** p < 0.01, ns = not significant (one-way ANOVA). The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Mass Spectrometry, Immunostaining, Control, Western Blot

    ( A, B ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that defects in proteostasis due to eff RNAi can be partially rescued by its human homolog UBE2D2, as indicated by the normalization of the detergent-soluble levels of poly-ubiquitinated proteins and p62/Ref(2)P. However, hUBE2D2 does not impact their detergent-insoluble levels, indicating that hUBE2D only partially rescues defects induced by UBE2D/eff RNAi. In (B), n = 3 (biological replicates), SEM, ** p < 0.01 (one-way ANOVA). ( C ) Muscle-specific UBE2D/eff RNAi reduces eff mRNA levels; n = 3 (biological replicates), SEM, ** p < 0.01 (one-way ANOVA). ( D ) Two distinct RNAi lines targeting UBE2D/eff reduce lifespan compared to control RNAi ( p < 0.001, log-rank test). ( E ) The decline in organismal survival due to UBE2D/eff RNAi in muscle is partially rescued by transgenic hUBE2D expression compared to control mcherry overexpression ( p < 0.001, log-rank test). The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: ( A, B ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that defects in proteostasis due to eff RNAi can be partially rescued by its human homolog UBE2D2, as indicated by the normalization of the detergent-soluble levels of poly-ubiquitinated proteins and p62/Ref(2)P. However, hUBE2D2 does not impact their detergent-insoluble levels, indicating that hUBE2D only partially rescues defects induced by UBE2D/eff RNAi. In (B), n = 3 (biological replicates), SEM, ** p < 0.01 (one-way ANOVA). ( C ) Muscle-specific UBE2D/eff RNAi reduces eff mRNA levels; n = 3 (biological replicates), SEM, ** p < 0.01 (one-way ANOVA). ( D ) Two distinct RNAi lines targeting UBE2D/eff reduce lifespan compared to control RNAi ( p < 0.001, log-rank test). ( E ) The decline in organismal survival due to UBE2D/eff RNAi in muscle is partially rescued by transgenic hUBE2D expression compared to control mcherry overexpression ( p < 0.001, log-rank test). The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Western Blot, Control, Transgenic Assay, Expressing, Over Expression

    ( A–C ) Proteasome activity assays from the thoraces of 14-day-old flies with muscle-specific knockdown of UBE2D/eff or mock, and with or without rescue by human UBE2D2. Measurement of the proteolytic activities of the proteasome indicates that UBE2D/eff RNAi significantly reduces the trypsin-like proteasome activity ( p = 0.0103), and there is also a trend for reducing the chymotrypsin-like activity ( p = 0.054). Expression of human UBE2D2 rescues the reduction in proteasome activity compared to a control transgene. The graphs display the mean ± SD with n = 5 (biological replicates). Statistical analysis was done with one-way ANOVA with Sidak’s multiple comparisons test; * p < 0.05. ( D–G ) Western blot analysis of detergent-soluble (D) and insoluble (E) fractions from the thoraces of 14-day-old flies with muscle-specific knockdown of UBE2D/eff or mock and with or without rescue by human UBE2D2. There are no significant changes in the soluble (F) and insoluble (G) levels of the 26S proteasome, as ascertained with antibodies raised against the p42A/Rpn7 component of the 19S regulatory particle (RP) and against the α subunits of the 20S catalytic core of the Drosophila proteasome. However, TMT mass spectrometry detects an average 15% increase in the levels of proteasome components . There are no significant changes in the total levels of Atg8, Atg8-I, Atg8-II, and Atg8-II/Atg8-I in the soluble fractions, indicating that UBE2D/eff does not substantially impact Atg8 levels and processing. There was, however, a significant decline in the insoluble Atg8-II/Atg8-I levels upon eff RNAi , which may indicate subtle changes in autophagy. The graphs display the mean ± SD with n = 5 (biological replicates). Statistical analysis was done with one-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01. The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: ( A–C ) Proteasome activity assays from the thoraces of 14-day-old flies with muscle-specific knockdown of UBE2D/eff or mock, and with or without rescue by human UBE2D2. Measurement of the proteolytic activities of the proteasome indicates that UBE2D/eff RNAi significantly reduces the trypsin-like proteasome activity ( p = 0.0103), and there is also a trend for reducing the chymotrypsin-like activity ( p = 0.054). Expression of human UBE2D2 rescues the reduction in proteasome activity compared to a control transgene. The graphs display the mean ± SD with n = 5 (biological replicates). Statistical analysis was done with one-way ANOVA with Sidak’s multiple comparisons test; * p < 0.05. ( D–G ) Western blot analysis of detergent-soluble (D) and insoluble (E) fractions from the thoraces of 14-day-old flies with muscle-specific knockdown of UBE2D/eff or mock and with or without rescue by human UBE2D2. There are no significant changes in the soluble (F) and insoluble (G) levels of the 26S proteasome, as ascertained with antibodies raised against the p42A/Rpn7 component of the 19S regulatory particle (RP) and against the α subunits of the 20S catalytic core of the Drosophila proteasome. However, TMT mass spectrometry detects an average 15% increase in the levels of proteasome components . There are no significant changes in the total levels of Atg8, Atg8-I, Atg8-II, and Atg8-II/Atg8-I in the soluble fractions, indicating that UBE2D/eff does not substantially impact Atg8 levels and processing. There was, however, a significant decline in the insoluble Atg8-II/Atg8-I levels upon eff RNAi , which may indicate subtle changes in autophagy. The graphs display the mean ± SD with n = 5 (biological replicates). Statistical analysis was done with one-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01. The data underlying the graphs shown in this figure can be found in the file. Uncropped western blots are available in .

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Activity Assay, Knockdown, Expressing, Control, Western Blot, Mass Spectrometry

    ( A, B ) TMT mass spectrometry of Drosophila skeletal muscle with UBE2D/eff knockdown vs. control RNAi (A) and overexpression of human UBE2D2 vs. control mcherry (B). The x -axis reports the log 2 FC, whereas the y -axis reports the–log 10 ( p -value). Examples of regulated proteins are shown in red. ( C ) Cross-comparison of the log 2 FC induced by eff RNAi vs. control RNAi ( x -axis) with the log 2 FC of hUBE2D2 vs. control mcherry ( y -axis). ( D ) Cross-comparison of the log 2 FC induced by eff RNAi + mcherry vs. control ( x -axis) with the log 2 FC of eff RNAi + hUBE2D2 vs. control ( y -axis) indicates that some of the protein changes induced by eff RNAi are rescued by hUBE2D2. ( E ) GO term analysis of protein categories that are enriched among up-regulated and down-regulated proteins in response to UBE2D/eff RNAi. The enrichment scores and number of regulated DAVID IDs are shown. ( F, G ) Examples of regulated proteins modulated by eff RNAi and hUBE2D-mediated rescue include protein changes that may drive derangement of proteostasis, such as Arc1/2 up-regulation (F), as well as protein changes that are protective and likely compensatory, such as proteasome components and chaperones (G); n = 3 (biological replicates), SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). See also and . The data underlying the graphs shown in this figure can be found in the file.

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: ( A, B ) TMT mass spectrometry of Drosophila skeletal muscle with UBE2D/eff knockdown vs. control RNAi (A) and overexpression of human UBE2D2 vs. control mcherry (B). The x -axis reports the log 2 FC, whereas the y -axis reports the–log 10 ( p -value). Examples of regulated proteins are shown in red. ( C ) Cross-comparison of the log 2 FC induced by eff RNAi vs. control RNAi ( x -axis) with the log 2 FC of hUBE2D2 vs. control mcherry ( y -axis). ( D ) Cross-comparison of the log 2 FC induced by eff RNAi + mcherry vs. control ( x -axis) with the log 2 FC of eff RNAi + hUBE2D2 vs. control ( y -axis) indicates that some of the protein changes induced by eff RNAi are rescued by hUBE2D2. ( E ) GO term analysis of protein categories that are enriched among up-regulated and down-regulated proteins in response to UBE2D/eff RNAi. The enrichment scores and number of regulated DAVID IDs are shown. ( F, G ) Examples of regulated proteins modulated by eff RNAi and hUBE2D-mediated rescue include protein changes that may drive derangement of proteostasis, such as Arc1/2 up-regulation (F), as well as protein changes that are protective and likely compensatory, such as proteasome components and chaperones (G); n = 3 (biological replicates), SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA). See also and . The data underlying the graphs shown in this figure can be found in the file.

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Mass Spectrometry, Knockdown, Control, Over Expression, Comparison

    (A–D) Cross-comparison of TMT mass spectrometry data from Drosophila skeletal muscle identifies substantial overlap in the proteomic changes induced by UBE2D/eff knockdown in young age compared to the changes that are induced by aging. The x -axis displays the significant ( p < 0.05) changes (log 2 FC) induced in skeletal muscle by eff RNAi ( Mhc>eff RNAi ) compared to control mcherry RNAi ( Mhc>mcherry RNAi ) at 2 weeks of age ( n = 3 biological replicates/group). The y -axis reports the significant ( p < 0.05) changes induced by aging in the skeletal muscle of control flies ( w 1118 ) when comparing 8 weeks (old) vs. 1 week (young), with n = 5 biological replicates/group. Among the proteins that are significantly regulated ( p < 0.05) by both UBE2D/eff knockdown and aging, approximately 70% are consistently regulated, i.e., either up-regulated (B, 51%) or down-regulated (A, 18.5%) by both, whereas the remaining approximately 30% is regulated oppositely by UBE2D/eff RNAi versus aging (A, D). The protein categories that are overrepresented in each group are indicated in (A–D) alongside the enrichment score. Representative proteins that are significantly regulated by UBE2D/eff RNAi in a consistent or discordant manner are shown in the graph and include Arc1 and Arc2. (E) Venn diagrams representing the overlap in the regulation of protein levels by aging and eff RNAi . These graphs were obtained from the list of significantly regulated proteins modulated by aging and eff RNAi . The threshold of log 2 FC>0.3 and <-0.3 was further applied for selecting up- and down-regulated proteins. The data underlying the graphs shown in this figure can be found in the file.

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: (A–D) Cross-comparison of TMT mass spectrometry data from Drosophila skeletal muscle identifies substantial overlap in the proteomic changes induced by UBE2D/eff knockdown in young age compared to the changes that are induced by aging. The x -axis displays the significant ( p < 0.05) changes (log 2 FC) induced in skeletal muscle by eff RNAi ( Mhc>eff RNAi ) compared to control mcherry RNAi ( Mhc>mcherry RNAi ) at 2 weeks of age ( n = 3 biological replicates/group). The y -axis reports the significant ( p < 0.05) changes induced by aging in the skeletal muscle of control flies ( w 1118 ) when comparing 8 weeks (old) vs. 1 week (young), with n = 5 biological replicates/group. Among the proteins that are significantly regulated ( p < 0.05) by both UBE2D/eff knockdown and aging, approximately 70% are consistently regulated, i.e., either up-regulated (B, 51%) or down-regulated (A, 18.5%) by both, whereas the remaining approximately 30% is regulated oppositely by UBE2D/eff RNAi versus aging (A, D). The protein categories that are overrepresented in each group are indicated in (A–D) alongside the enrichment score. Representative proteins that are significantly regulated by UBE2D/eff RNAi in a consistent or discordant manner are shown in the graph and include Arc1 and Arc2. (E) Venn diagrams representing the overlap in the regulation of protein levels by aging and eff RNAi . These graphs were obtained from the list of significantly regulated proteins modulated by aging and eff RNAi . The threshold of log 2 FC>0.3 and <-0.3 was further applied for selecting up- and down-regulated proteins. The data underlying the graphs shown in this figure can be found in the file.

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Comparison, Mass Spectrometry, Knockdown, Control

    The ubiquitin-conjugating enzyme UBE2D/eff has a key role in proteostasis in skeletal muscle by maintaining optimal proteasome activity. UBE2D/eff protein levels decline during aging. Experimentally reducing UBE2D/eff levels from a young age causes a loss in protein quality control, a consequent precocious surge in the levels of insoluble poly-ubiquitinated proteins (which normally accumulate only in old age because of age-associated defects in proteostasis), and shortens lifespan. Proteomics surveys indicate that UBE2D/eff knockdown induces proteomic changes similar to aging, and that such UBE2D/eff RNAi -induced changes are rescued by transgenic expression of its human homolog UBE2D2. Some of the proteins that are up-regulated by aging and UBE2D/eff RNAi include proteostasis regulators (such as chaperones and Pomp) that are transcriptionally induced as part of an adaptive stress response to the decline in proteostasis. Altogether, these findings indicate that UBE2D/eff is necessary to maintain a youthful proteome and to ensure muscle protein quality control during aging by sustaining proteasome activity.

    Journal: PLOS Biology

    Article Title: The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity

    doi: 10.1371/journal.pbio.3002998

    Figure Lengend Snippet: The ubiquitin-conjugating enzyme UBE2D/eff has a key role in proteostasis in skeletal muscle by maintaining optimal proteasome activity. UBE2D/eff protein levels decline during aging. Experimentally reducing UBE2D/eff levels from a young age causes a loss in protein quality control, a consequent precocious surge in the levels of insoluble poly-ubiquitinated proteins (which normally accumulate only in old age because of age-associated defects in proteostasis), and shortens lifespan. Proteomics surveys indicate that UBE2D/eff knockdown induces proteomic changes similar to aging, and that such UBE2D/eff RNAi -induced changes are rescued by transgenic expression of its human homolog UBE2D2. Some of the proteins that are up-regulated by aging and UBE2D/eff RNAi include proteostasis regulators (such as chaperones and Pomp) that are transcriptionally induced as part of an adaptive stress response to the decline in proteostasis. Altogether, these findings indicate that UBE2D/eff is necessary to maintain a youthful proteome and to ensure muscle protein quality control during aging by sustaining proteasome activity.

    Article Snippet: Interestingly, the proteostasis deficits caused by UBE2D/eff knockdown are partially rescued by transgenic expression of one of its human homologs, UBE2D2, suggesting that UBE2D ubiquitin-conjugating enzymes have evolutionary conserved roles in maintaining protein quality control during aging.

    Techniques: Ubiquitin Proteomics, Activity Assay, Control, Knockdown, Transgenic Assay, Expressing