Journal: Cell reports

Article Title: MYO10 regulates genome stability and cancer inflammation through mediating mitosis

doi: 10.1016/j.celrep.2023.112531

Figure Lengend Snippet:

Article Snippet: Anti-UbcH7 , Bio-Techne/NOVUS Biologicals , #NB100-2265, rabbit polyclonal, RRID: AB_2304026.

Techniques: Staining, Recombinant, SYBR Green Assay, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Knock-Out, Software, Fluorescence, Microscopy

Journal: Leukemia

Article Title: Distinct characteristics of VEXAS-causative UBA1 M41 and recurrent functional non-M41 mutations

doi: 10.1038/s41375-025-02775-4

Figure Lengend Snippet: A Schematic representation of the UBA1 gene, highlighting its functional domains and pinpointing the exact locations of the identified UBA1 variants. The variants were classified into Tier-1 (pathogenic: P), Tier-2 (likely pathogenic: LP) and Tier-3 (variant of uncertain significance: VUS), as depicted in the outer pie chart on the left. UBA1 variants are displayed on top of the gene if the locus occurred recurrently in our screen. Combination of variants detected in the same patients are also shown. Variants detected only as combinations are italicized. M41 and other tier assignments are color-coded. Bold underlined variants were tested for functional significance. B Chart summarizing UBA1 variants and their functional status, indicating the presence or absence of ubiquitylation. Quantification of polyubiquitin levels and mono-ubiquitylated histone H2A/B (Ub-H2A, Ub-H2B) or E2 enzymes (UBE2D3-Ub, UBE2L3-Ub) were normalized within each sample to β-actin levels and scaled to WT transfection. E2 enzyme ubiquitylation was quantified as the ratio of charged form to uncharged form and scaled to WT. Data represent n = 3–6 biological replicates, shown as mean −/+ s.d., significance determined by unpaired t-test with Welch’s correction (*p < 0.05, **p < 0.01).

Article Snippet: Primary antibodies for Poly-ubiquitin (Cell Signaling, 3936S), UBE2D3 (Cell-Signaling, 4330), UBE2L3 (R&D Systems, E2-640), ubiquitylated-H2A (Cell Signaling, 8240), H2A (Cell Signaling, 12349), ubiquitylated-H2B (Cell Signaling, 5546S), H2B (Cell Signaling, 12364), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:3000.

Techniques: Functional Assay, Variant Assay, Transfection

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: rs140490 Is Associated with SLE and a Cell-Specific Increase in UBE2L3 Production (A) Locuszoom plot showing SNPs around UBE2L3 imputed to 1000 Genomes level. Recombination rate was calculated from HapMap data. (B) rs140490 is associated with increased expression in microarray data (expressed as log base 2, bars show median and interquartile range) from EBV-transformed lymphoblastoid cell lines, as well as increased mRNA expression in CD19 + B cells and CD14 + monocytes isolated from PBMCs. (C) Semiquantitative analysis of UBE2L3 protein level from immunoblot densitometry compared to actin, stratified by rs140490 genotype in CD19 + B cells and CD4 + T cells isolated from PBMCs from healthy individuals, with representative immunoblots shown from individuals for each rs140490 genotype.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Expressing, Microarray, Transformation Assay, Isolation, Western Blot

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: UBE2L3 Exerts Rate-Limiting Control over LUBAC-Mediated NF-κB Activation (A) Basal NF-κB luciferase activity in HEK293 cells in response to increased production of combinations of HOIL-1, HOIP, Sharpin, and UBE2L3. UBE2L3 enhances NF-κB activation due to increased levels of HOIL-1 and HOIP. Error bars represent standard error of the mean. (B) Dominant-negative variant p.Cys86Ser UBE2L3 suppresses NF-κB activity due to LUBAC. (C) UBE2L3 is the preferred E2 ubiquitin-conjugating enzyme for LUBAC in vivo. Luciferase assay time course over 48 hr in HEK293-NF-κB reporter cells transiently transfected different E2 ubiquitin-conjugating enzymes in combination with EV, HOIP alone, HOIL-1+HOIP, or Sharpin+HOIP, comparing the basal NF-κB response after increased production of individual E2 enzymes with LUBAC. (D and E) Comparison of NF-κB p65 translocation measured by Imagestream analysis in HEK293 cells transiently transfected with empty vector (EV) or HOIL-1+V5-HOIP+UBE2L3. p65 nuclear translocation is quantified by Imagestream similarity feature correlating fluorescence co-localization of AlexaFluor488-p65 with nuclear DAPI. Similarity histograms show levels of p65 translocation in EV, compared to unstimulated HEK293 cells transfected with HOIL-1+HOIP+UBE2L3. EV-transfected cells stimulated with TNF 10 ng/ml for 1 hr acted as a positive control. AlexaFluor647 anti-V5-tag and PE anti-UBE2L3 were used to gate cells with high levels of HOIP(V5) and UBE2L3, showing high p65 translocation in this group compared to cells with normal HOIP and UBE2L3 levels. Representative histograms are shown from one of three separate experiments. (D) Representative Imagestream images of HEK293 cells with median similarity level of p65 translocation as measured in (E) showing AlexaFluor488-p65 (green), DAPI nuclear dye (pseudocolored red), PE-UBE2L3 (yellow), and AlexaFluor647-V5(HOIP) (purple). Merged images show p65/DAPI overlap in yellow.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Control, Activation Assay, Luciferase, Activity Assay, Dominant Negative Mutation, Variant Assay, Ubiquitin Proteomics, In Vivo, Transfection, Comparison, Translocation Assay, Plasmid Preparation, Fluorescence, Positive Control

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: UBE2L3 and LUBAC Augment Late-Phase NF-κB Activation in Response to TNF (A) Increased production of LUBAC in combination with UBE2L3 augments NF-κB activation after 10 hr of TNF stimulation. This augmentation of the TNF response is abrogated by variant p.Cys86Ser UBE2L3 or variant p.Cys885Ser HOIP. (B) Time course showing NF-κB activation after TNF stimulation in response to increased synthesis of LUBAC and UBE2L3. Maximal dysregulation of NF-κB occurs in the late-phase response to TNF. Error bars represent standard error of the mean.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Activation Assay, Variant Assay

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: Inhibition of UBE2L3 Regulates NF-κB Signaling and Target Gene Transcription (A) Luciferase assay in HEK293-NF-κB reporter cell line showing that siRNA inhibition of HOIL-1, HOIP, Sharpin, and UBE2L3 inhibits NF-κB activation in response to TNF. Error bars represent standard error of the mean. (B) Immunoblot showing siRNA knockdown of HOIL-1, HOIP, Sharpin, and UBE2L3. (C) Inhibition of UBE2L3 or HOIP in HEK293 cells leads to basal increase in IκBα levels and impaired phosphorylation of IκBα in response to TNF, and subsequent IκBα degradation is reduced. UBE2L3 does not impair JNK or ERK phosphorylation. (D) Inhibition of UBE2L3 or HOIP reduces transcription of NF-κB target genes measured by real-time qPCR. HEK293 cells transfected with control, UBE2L3, or HOIP siRNA were stimulated with 10 ng/ml TNF.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Inhibition, Luciferase, Activation Assay, Western Blot, Knockdown, Phospho-proteomics, Transfection, Control

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: UBE2L3 Genotype Affects Basal and Stimulated NF-κB Translocation in Primary Human B Cells and Monocytes Translocation of NF-κB p65 was quantified by similarity score on Imagestream analysis of CD19 + B cells and CD14 + monocytes isolated from PBMCs from healthy individuals, stratified by genotype at rs140490. (A) Representative similarity histograms measuring co-localization of p65 and nuclear dye DRAQ5 for B cells after 30 min of stimulation with CD40L and for monocytes after 30 min of TNF stimulation for each rs140490 genotype. Translocated cells were defined as similarity score > 1.5. (B) Imagestream images for each rs140490 genotype showing CD40L-stimulated B cells and TNF-stimulated monocytes of median similarity levels of p65 translocation from histograms in (A) showing p65 (Alexa488), DRAQ5 nuclear dye, and merged images. (C) Graphs show percent of NF-κB p65 nuclear translocated cells in unstimulated CD19 + B cells and CD14 + monocytes from genotyped individuals. (D) Graphs show 60 min time course of p65 translocation in response to stimulation of B cells with 0.1 μg/ml CD40L and monocytes with 10 ng/ml TNF, stratified by rs140490 genotype. Error bars represent standard error of the mean.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Translocation Assay, Isolation

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: UBE2L3 Is Highly Abundant in Peripheral Blood Plasmablasts and Plasma Cells PBMCs isolated from healthy individuals and SLE-affected individuals were subject to ten-color flow cytometry to analyze UBE2L3 levels in B cell subsets. (A) Gating strategy for B cell subsets. (B) Histograms of B cell subsets from a representative SLE-affected individual demonstrating clear shift in UBE2L3 protein levels in CD19 mid CD20 − CD27 hi CD38 hi IgD − plasmablast and CD20 − CD27 hi CD38 hi IgD − CD138 hi plasma cell populations (isotype control light gray). (C) UBE2L3 abundance was highly increased in plasmablasts and plasma cells compared to other B cell subsets in both SLE-affected individuals and controls. (D and E) UBE2L3 levels were significantly higher in (D) Ki-67 + proliferating B cells and (E) CD95 + activated B cells in SLE-affected individuals. Histograms: isotype control, light gray; blue, Ki-67 − or CD95 − ; red, Ki-67 + or CD95 + . Error bars represent standard error of the mean.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Clinical Proteomics, Isolation, Flow Cytometry, Control

Journal: American Journal of Human Genetics

Article Title: UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

doi: 10.1016/j.ajhg.2014.12.024

Figure Lengend Snippet: UBE2L3 Genotype Influences Peripheral Blood Plasmablast Proliferation in SLE (A) The UBE2L3 risk allele (rs140490 genotype) was associated with a significant expansion of peripheral blood plasmablasts and plasma cells (expressed as percent of live B cells) from SLE-affected individuals (n = 29), whereas this expansion was not seen in healthy controls (n = 25). Error bars represent standard error of the mean. (B) Representative flow cytometry plots showing percentages of plasmablasts and plasma cells (expressed as percent of live B cells) from SLE-affected individuals for each homozygous genotype.

Article Snippet: The following plasmids on the pCMV6 vector were purchased from Origene: untagged UBE2L3, HOIP, UBE2D1, UBE2D2, UBE2D3, Sharpin-Myc-DDK, and empty vector (pCMV6-AC). pCMV6-HOIL-1-Myc-DDK (Origene) was subcloned into pCMV6AC to remove the C terminus Myc-DDK tag. pcDNA3.1-V5-His-HOIP and pcDNA3.1-V5-His-HOIP (p.Cys885Ser) were kind gifts of H. Walczak. pFlagCMV2-UbcH8 (UBE2L6) was obtained from Addgene.

Techniques: Clinical Proteomics, Flow Cytometry

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Overall Structure of FANCL-Ube2T Complex (A) The overall structure of the RING domain of FANCL (magenta) bound to Ube2T (blue) is shown in cartoon representation. Gray spheres represent zinc ions. A gold star represents the position of Ube2T’s catalytic cysteine. (B) RING domain of FANCL (magenta) overlain with c-cbl RING domain (green; PDB ID code 1FBV ). (C) Ube2T (blue) overlain with Ube2L3 (orange; PDB ID code 1FBV ) showing the structural conservation of the UBC fold, comprising a four-stranded β-meander flanked by an N-terminal helix (helix1) and two C-terminal helices (helixes 2 and 3). A gold star represents the position of the catalytic cysteine. The gray oval shows the E3 binding interface of E2s. (D) Top left panel: The pi stacking in the binding interface between Y311 of FANCL and R6 and R9 of Ube2T. Top right panel: The hydrophobic binding interface of the RING domain (magenta) and Ube2T (blue). Bottom panels: The electrostatic and hydrogen bonding network of the RING-Ube2T interface. Interactions are represented by dashed lines. See also Figure S1 .

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Binding Assay

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Structural Comparison of the FANCL RING Domain-Ube2t Complex with Other RING-E2 Complexes (A) A structure-based sequence alignment of E2s. PDB ID codes of E2s, as listed in the figure: 1FBV , 3RPG , 4AP4 , 4AUQ , 3RZ3 , 2YB6 , 3K9O , 3H8K , 2Z5D , 2F4W , 3HCT , and 3BZH . (B) A structure-based sequence alignment of RING and Ubox domains. Ubox domains are highlighted by a cyan box. PDB ID codes used of RING and Ubox domains, as listed in the figure: 1FBV , 4F52 , 4AUQ , 3HCT , 2C2V , 3LIZ , 3RPG , 4AP4 , 4EPO , 2YHO , 2Y43 , 4KBL , and 4K7D . Residues shaded in red to yellow colors indicate conserved residues, where red corresponds to strict conservation. Gray bars indicate zinc coordinating atoms. Green circles highlight residues involved in the hydrophobic interface between FANCL and Ube2T. Purple circles denote residues involved in hydrogen bonding and electrostatic interactions in the FANCL Ube2T interface. (C) Superpositions of the FANCL RING-Ube2T complex (colored pink and blue, respectively), with c-cbl RING-Ube2L3 complex (left) shaded gray (PDB ID code 1FBV ), idol-Ube2D1 complex (middle) shaded gray (PDB ID code 2YHO ), and ring1b-Ube2D3 complex (right) shaded gray (PDB ID code 3RPG ). Numbered residues are the same as the FANCL RING-Ube2T complex, with dashed lines showing interactions.

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Sequencing

Journal: Structure(London, England:1993)

Article Title: Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

doi: 10.1016/j.str.2013.12.004

Figure Lengend Snippet: Conserved Hydrophobic RING Residues Are Required for Ube2T Binding and FANCL Selects Solely Ube2T In Vitro (A) Size-exclusion chromatogram profiles of wild-type (WT) or mutant RING domains (green dashed line) and WT Ube2T (blue dotted line) overlaid with profiles from binding experiments in which WT Ube2T has been incubated with WT or mutant RING domains (pink line) and subjected to size-exclusion chromatography. Binding was assessed by complex formation, which is indicated by a peak shift to the left labeled complex. (B) Size-exclusion chromatogram of FANCL RING domain incubated with an E2 mix consisting of Ube2T, Ube2D3, and Ube2L3 (pink line). Chromatograms of Ube2T (blue dotted line) and the RING domain (green dashed line) are also overlaid. A peak shift to the left is observed, indicating complex formation. SDS-PAGE gel of the fractions collected from the size-exclusion experiment and stained with Coomassie Brilliant Blue. The E2 gel bands found in the shifted peak were assessed by mass spectrometry for protein identification and confirmed as exclusively Ube2T. See also Figure S2 .

Article Snippet: Human Ube2D3 (UbcH5c), Ube2L3 (UbcH7), Ube2K, Ube2B, and Ube2R1 were cloned from I.M.A.G.E. clones (Geneservice) and inserted into the pDEST17 (Invitrogen) and pET RSF vectors containing an N-terminal 6xHis tag and a TEV cleavage site.

Techniques: Binding Assay, In Vitro, Mutagenesis, Incubation, Size-exclusion Chromatography, Labeling, SDS Page, Staining, Mass Spectrometry