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(A) Schematic of human TRIM32 highlighting domain architecture. The four lysine residues in the coiled-coil domain identified by mass spectrum analysis as autoubiquitination sites in the current study are indicated in red. The hydrophobic helix loop in TRIM32 NHL domain is labeled in blue. (B) Depiction of the core components of the TRIM32 in vitro auto-ubiquitination reconstitution assay including UBE1, the E2 enzyme <t>UBE2D3,</t> ubiquitin, TRIM32, and ATP. (C) Coomassie stained SDS-PAGE gels of in vitro TRIM32 autoubiquitination assays showing that compared with WT TRIM32, the quadruple point mutation TRIM32 4KR (K175R/K182R/K204R/K215R) prevents autoubiquitination. Colored box indicates the band for TRIM32 (cyan) and ubiquitin (blue) level quantification, respectively. Red arrow indicates HWM polyubiquitinated TRIM32 in the loading well. (D) Quantification of time course change in (C) measuring TRIM32 or ubiquitin level via densitometry. Combined data from three independent experiments are shown. Data are presented as mean ± s.e.m. (E) Ubiquitin linkage composition by AQUA MS from in vitro TRIM32 autoubiquitination assays performed in technical duplicate. (F) Coomassie stained SDS-PAGE gel image representing TRIM32 autoubiquitination assays before and after Lb pro * treatment for 1 hours. Released ubiquitin (blue box) was analyzed by MS to identify GG-modified ubiquitin species. (G) TRIM32 assembles branched polyUb, according to intact MS analysis. Quantification by spectra deconvolution of individual ubiquitin species from (F) in autoubiquitination assay.

Ube2d3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ubch5c+wt+pet28a/UbcH5c+WT+pET28a+(Plasmid+%2312643)/bio_rxiv__64898__2026__02__11__705390-208-11-12
Average 93 stars, based on 20 article reviews

ube2d3 - by Bioz Stars, 2026-09

93/100 stars

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1) Product Images from "TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates"

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

Journal: bioRxiv

doi: 10.64898/2026.02.11.705390

Figure Legend Snippet: (A) Schematic of human TRIM32 highlighting domain architecture. The four lysine residues in the coiled-coil domain identified by mass spectrum analysis as autoubiquitination sites in the current study are indicated in red. The hydrophobic helix loop in TRIM32 NHL domain is labeled in blue. (B) Depiction of the core components of the TRIM32 in vitro auto-ubiquitination reconstitution assay including UBE1, the E2 enzyme UBE2D3, ubiquitin, TRIM32, and ATP. (C) Coomassie stained SDS-PAGE gels of in vitro TRIM32 autoubiquitination assays showing that compared with WT TRIM32, the quadruple point mutation TRIM32 4KR (K175R/K182R/K204R/K215R) prevents autoubiquitination. Colored box indicates the band for TRIM32 (cyan) and ubiquitin (blue) level quantification, respectively. Red arrow indicates HWM polyubiquitinated TRIM32 in the loading well. (D) Quantification of time course change in (C) measuring TRIM32 or ubiquitin level via densitometry. Combined data from three independent experiments are shown. Data are presented as mean ± s.e.m. (E) Ubiquitin linkage composition by AQUA MS from in vitro TRIM32 autoubiquitination assays performed in technical duplicate. (F) Coomassie stained SDS-PAGE gel image representing TRIM32 autoubiquitination assays before and after Lb pro * treatment for 1 hours. Released ubiquitin (blue box) was analyzed by MS to identify GG-modified ubiquitin species. (G) TRIM32 assembles branched polyUb, according to intact MS analysis. Quantification by spectra deconvolution of individual ubiquitin species from (F) in autoubiquitination assay.

Techniques Used: Labeling, In Vitro, Ubiquitin Proteomics, Reconstitution Assay, Staining, SDS Page, Mutagenesis, Modification

Plasmid Preparation:

Article Title: Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity
Article Snippet: .. UbcH5c WT pET28a (plasmid 12643) and UbcH5c S22R pET28a (plasmid 12644) ( 15 ) were obtained from Addgene. .. The following reagents were purchased from Boston Biochem: ubiquitin E1 (E-305), UbcH5a (E2–616), Mg 2+ -ATP (B-20), ubiquitin (U-100H), methylated ubiquitin (U-501), Lys 48 -only ubiquitin (UM-K480), and I44A ubiquitin (UM-I44A).

Article Title: Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates
Article Snippet: .. UbcH5c WT pET28a was a gift from Rachel Klevit (Addgene plasmid # 12643; http://n2t.net/addgene:12643 ; RRID:Addgene_12643) . pET3a-hUBA1 was a gift from Titia Sixma (Addgene plasmid # 63571; http://n2t.net/addgene:63571 ; RRID:Addgene_63571) . .. To generate a baculovirus expression vector of a monomeric EGFP-CBX8 (mEGFP-CBX8) construct, first GFP was amplified from a pSpCas9(BB)-2A-GFP vector and CBX8 was amplified from a pFB1.HMBP.A3.PrS.ybbR vector containing CBX8 as an insert.

Article Title: Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.
Article Snippet: .. UbcH5c WT pET28a was a gift from Rachel Klevit (Addgene plasmid #12643; http://n2t.net/addgene:12643; RRID: Addgene_12643)51. pET3a-hUBA1 was a gift from Titia Sixma (Addgene plasmid # 63571; http://n2t.net/addgene:63571; RRID:Addgene_63571)52. .. To generate a baculovirus expression vector of a monomeric enhanced GFP (EGFP)–CBX8 (mEGFP–CBX8) construct, first GFP was amplified from a pSpCas9(BB)-2A-GFP vector and CBX8 was amplified from a pFB1.HMBP.A3.PrS.ybbR vector containing CBX8 as an insert.

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates
Article Snippet: .. UbcH5c WT pET28a was a gift from Rachel Klevit (Addgene plasmid #12643; http://n2t.net/addgene:12643 ; RRID: Addgene_12643 ) . pET3a-hUBA1 was a gift from Titia Sixma (Addgene plasmid # 63571; http://n2t.net/addgene:63571 ; RRID: Addgene_63571 ) . .. To generate a baculovirus expression vector of a monomeric enhanced GFP (EGFP)–CBX8 (mEGFP–CBX8) construct, first GFP was amplified from a pSpCas9(BB)-2A-GFP vector and CBX8 was amplified from a pFB1.HMBP.A3.PrS.ybbR vector containing CBX8 as an insert.

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Journal: bioRxiv

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

doi: 10.64898/2026.02.11.705390

Figure Lengend Snippet: (A) Schematic of human TRIM32 highlighting domain architecture. The four lysine residues in the coiled-coil domain identified by mass spectrum analysis as autoubiquitination sites in the current study are indicated in red. The hydrophobic helix loop in TRIM32 NHL domain is labeled in blue. (B) Depiction of the core components of the TRIM32 in vitro auto-ubiquitination reconstitution assay including UBE1, the E2 enzyme UBE2D3, ubiquitin, TRIM32, and ATP. (C) Coomassie stained SDS-PAGE gels of in vitro TRIM32 autoubiquitination assays showing that compared with WT TRIM32, the quadruple point mutation TRIM32 4KR (K175R/K182R/K204R/K215R) prevents autoubiquitination. Colored box indicates the band for TRIM32 (cyan) and ubiquitin (blue) level quantification, respectively. Red arrow indicates HWM polyubiquitinated TRIM32 in the loading well. (D) Quantification of time course change in (C) measuring TRIM32 or ubiquitin level via densitometry. Combined data from three independent experiments are shown. Data are presented as mean ± s.e.m. (E) Ubiquitin linkage composition by AQUA MS from in vitro TRIM32 autoubiquitination assays performed in technical duplicate. (F) Coomassie stained SDS-PAGE gel image representing TRIM32 autoubiquitination assays before and after Lb pro * treatment for 1 hours. Released ubiquitin (blue box) was analyzed by MS to identify GG-modified ubiquitin species. (G) TRIM32 assembles branched polyUb, according to intact MS analysis. Quantification by spectra deconvolution of individual ubiquitin species from (F) in autoubiquitination assay.

Article Snippet: Bacterial plasmids obtained from Addgene include: UBE1 (Addgene plasmid # 34965), UBE2D3 (Addgene plasmid # 12643), ANXA11 (Addgene plasmid # 164496), p62 (Addgene plasmid # 190929), TDP-43 (Addgene plasmid # 133320 and plasmid # 27462).

Techniques: Labeling, In Vitro, Ubiquitin Proteomics, Reconstitution Assay, Staining, SDS Page, Mutagenesis, Modification

a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

Journal: Nature Structural & Molecular Biology

Article Title: Dynamic PRC1–CBX8 stabilizes a porous structure of chromatin condensates

doi: 10.1038/s41594-024-01457-6

Figure Lengend Snippet: a , MNase digestion of reconstituted Chromatin and a naked DNA (same sequence as used for chromatin reconstitution). DNA fragments post digestion are resolved on a 1.2 % Agarose gel. Protected bands indicating mono- and di-nucleosome core particles (NCP and diNCP) are indicated by the arrows. b , 3 ug of each protein complex used in this study resolved on a 4–12% SDS-PAGE gel stained with Coomassie. c , Ubiquitylation activity of each protein complex used in this study visualized on a western blot. All samples include UBA1, UBCH5C, Ubiquitin, ATP and 1 μM chromatin (nucleosome concentration). Blot is representative of two independent experiments. d , Phase separation experiment comparing chromatin condensation activity of PRC1 C8 with MBP-tagged CBX8 to PRC1 C8 with the tag cleaved.

Article Snippet: UbcH5c WT pET28a was a gift from Rachel Klevit (Addgene plasmid #12643; http://n2t.net/addgene:12643 ; RRID: Addgene_12643 ) . pET3a-hUBA1 was a gift from Titia Sixma (Addgene plasmid # 63571; http://n2t.net/addgene:63571 ; RRID: Addgene_63571 ) .

Techniques: Sequencing, Agarose Gel Electrophoresis, SDS Page, Staining, Activity Assay, Western Blot, Ubiquitin Proteomics, Concentration Assay

$Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 μL), cell pellet (P) (6 μL), cell lysate supernatant (S) (3 μL), Ni-NTA resin flowthrough (FT) (3 μL), and Ni-NTA elution fractions (lanes 6–11) (3 μL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (D) SDS-PAGE of purified E2s (UBE2D3, UBE2W, UBE2N, UBE2E3, UBE2V2, UBE2E2, UBE2E1, UBE2B), Lane 1 shows MW markers.$

Journal: STAR Protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: Purification of UBE2D3 and other E2s (A) Whole cell lysate (WCL) (6 μL), cell pellet (P) (6 μL), cell lysate supernatant (S) (3 μL), Ni-NTA resin flowthrough (FT) (3 μL), and Ni-NTA elution fractions (lanes 6–11) (3 μL) of UBE2D3 were analyzed by SDS-PAGE and Coomassie staining. (B) The UBE2D3 elution fractions from SP cation affinity chromatography (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (C) The UBE2D3 elution fractions from size exclusion chromatography (SEC) (3 μL) were analyzed by SDS-PAGE and Coomassie staining. (D) SDS-PAGE of purified E2s (UBE2D3, UBE2W, UBE2N, UBE2E3, UBE2V2, UBE2E2, UBE2E1, UBE2B), Lane 1 shows MW markers.

Article Snippet: The plasmids of pFastbac-Flag-BRCA1 (modified based on the plasmid from Jeffrey Parvin; deposited in Addgene (#223228)) and pFastBac-TwinStrepTag-BARD1 (Addgene#137166) are used for Flag-BRCA1 and Twin-StrepTag-BARD1 expression in insect cells., c. The expression plasmids for human E1 (pET3a-hUBA1 (Addgene#63571)), E2s (pET28a-UBE2D3 (Addgene#12643), pET15-UBE2W, pET24a-UBE2E3, pET24a-UBE2E2, pET24a-UBE2E1, pET24a-UBE2N, pET24a-UBE2V2), and Ubiquitin (pET15-Ub (Addgene#12647)) were described before., , , Plasmids of pDEST17-UBE2B (Addgene#15781) was from Addgene.

Techniques: Purification, SDS Page, Staining, Affinity Chromatography, Size-exclusion Chromatography

In vitro ubiquitylation of histone H2A by BRCA1-BARD1 and its variants (A) Schematic of ubiquitination assay mediated by E3 BRCA1-BARD1 with NCP as a substrate. (B) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency of BRCA1-BARD1 in conjunction with different E2s. UBE2B that does not bind BRCA1-BARD1 was included as a negative control. BC1-BD1, BRCA1-BARD1. (C) The BRCA1-BARD1 RING structure showing the locations of BRCA1 residues I26, L63, and K65 with the E2 binding surface indicated by dotted lines (PDB: 7JZV ). (D) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency for wild-type or mutant BRCA1-BARD1 in conjunction with NCP as a substrate and E2s: UBE2D3 (top) and UBE2D3 + UBE2N-UBE2V2 (bottom).

Journal: STAR Protocols

Article Title: Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation

doi: 10.1016/j.xpro.2024.103294

Figure Lengend Snippet: In vitro ubiquitylation of histone H2A by BRCA1-BARD1 and its variants (A) Schematic of ubiquitination assay mediated by E3 BRCA1-BARD1 with NCP as a substrate. (B) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency of BRCA1-BARD1 in conjunction with different E2s. UBE2B that does not bind BRCA1-BARD1 was included as a negative control. BC1-BD1, BRCA1-BARD1. (C) The BRCA1-BARD1 RING structure showing the locations of BRCA1 residues I26, L63, and K65 with the E2 binding surface indicated by dotted lines (PDB: 7JZV ). (D) Representative nucleosome ubiquitylation assays monitoring H2A-Ub efficiency for wild-type or mutant BRCA1-BARD1 in conjunction with NCP as a substrate and E2s: UBE2D3 (top) and UBE2D3 + UBE2N-UBE2V2 (bottom).

Techniques: In Vitro, Ubiquitin Proteomics, Negative Control, Binding Assay, Mutagenesis

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ube2d3 (Addgene inc)

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1) Product Images from "TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates"

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