Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: PD-L1 is modified by UBL3. ( a ) Co-immunoprecipitation (IP) analysis of using MDA-MB-231 cell lysates expressing Flag-tagged PD-L1 (PD-L1-Flag) and biotinylated-tagged UBL3 (Biotin-UBL3). ( b ) Co-IP was performed using cell lysates expressing PD-L1-Flag or PD-L1 mutants-Flag (C250A, C272A, C250/272A) along with Biotin-UBL3 in MDA-MB-231 cells. IB analysis was conducted using the indicated antibodies. ( c ) the relative intensity of UBL3 modification (Flag/SA-HRP) calculated as the ratio of UBL3-modified PD-L1 signals (> 70 kDa) to input SA-HRP signals, normalized to the mean of cells transfected with Biotin-UBL3 and wild-type PD-L1. Data are presented as mean ± s.e.m., with dots representing individual experiments. One-way ANOVA with Tukey’s multiple comparisons test. ns, not significant, * P < 0.05, ** P < 0.001, *** P < 0.0005, PD-L1 vs. C250A, P = 0.1708; PD-L1 vs. C272A, P = 0.0009; PD-L1 vs. C250/272A, P = 0.0002; C250A vs. C272A, P = 0.0135; C272A vs. C250/272A, P = 0.5847; TMD, transmembrane domain; Flag, Flag-tag; βME-, without 2-mercaptoethanol; βME+, with 2-mercaptoethanol; SA-HRP, streptavidin-horseradish peroxidase.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Modification, Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection, FLAG-tag

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 regulates PD-L1 levels in sEVs. ( a ) Immunoblot (IB) analysis. The cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors were blotted with various antibodies. ( b ) IB analysis. The cell lysates and sEVs from the stable UBL3 knockdown H1299 cell lines were blotted with various antibodies. CD9 and CD63 were used as sEV markers, as they are tetraspanin proteins abundantly expressed on the sEV surface . Relative intensity of PD-L1 in sEVs was calculated as PD-L1/CD63 and presented as fold change, normalized to the mean value of the respective control (mock for a; shNega for b). Data are presented as mean ± s.e.m., with dots representing individual experiments. Two-tailed unpaired t-test. *** P < 0.005.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Western Blot, Transfection, Knockdown, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: Inhibition of UBL3 modification and PD-L1 sorting by statins. ( a and b ), Effect of pitavastatin treatment on UBL3 modification in WM9 ( a ) and H1299 ( b ) cells. Five hours after transfection, 10 µM pitavastatin was added. ( c and d ), IB analysis of the cell lysates and sEVs from the conditioned medium of H1299 cells transfected with 3xFlag-UBL3 vectors ( c ) or untransfected ( d ) were blotted with various antibodies. Five hours after gene transfection, 0.2 µM pitavastatin was added. Pit, Pitavastatin. Right panels, the relative intensity of PD-L1 in sEVs (c: PD-L1/CD63 [upper] normalized to the mean of mock − untreated control, and UBL3/CD63 [lower] normalized to the mean of Flag-UBL3 − untreated control; d: PD-L1/CD63 [upper] and UBL3/CD63 [lower], both normalized to the mean of untreated control). Data are presented as mean ± s.e.m., with dots representing individual experiments. As inhibition of UBL3 modification was observed even at low concentrations of pitavastatin (Supplementary Fig. S4b), a low concentration of pitavastatin was used in sEV purification experiments. IB analysis using the indicated antibodies. Flag, Flag-tag; GAPDH, internal control; CD63 and CD9, sEVs markers; PD-L1, endogenous PD-L1; UBL3, endogenous UBL3. βME−, without 2-mercaptoethanol. βME+, with 2-mercaptoethanol. ( c ), one-way ANOVA with Tukey’s multiple comparisons test. ( d ), Two-tailed unpaired t-test. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Inhibition, Modification, Transfection, Control, Concentration Assay, Purification, FLAG-tag, Two Tailed Test

Journal: Scientific Reports

Article Title: Statins attenuate PD-L1 sorting to small extracellular vesicles dependent on ubiquitin-like 3 modification

doi: 10.1038/s41598-025-27789-x

Figure Lengend Snippet: UBL3 and PD-L1 expression levels influence survival in lung cancer patients. ( a ) Kaplan–Meier survival curves for the lung squamous cell carcinoma cohort, stratified by UBL3 and PD-L1 expression. ( b ) Survival curves for the same cohort, stratified by UBL3 and PD-1 expression. Hazard ratios and p-values from log-rank (Mantel–Cox) tests are summarized in Supplementary Figure S7a.

Article Snippet: The antibodies used in the study were as follows: UBL3 (1:100; lab-generated; 1:1000; 14100-1-AP [Lot#00005139], Proteintech), Flag (1:1000; F3165, Sigma), streptavidin-HRP (1:5000; 19534-050, Invitrogen), GAPDH (1:1000; 2118, Cell Signaling), CD63 (1:1000; Ts63, Thermo), CD9 (1:1000; Ts9, Thermo), PD-L1 (1:500; 405.9A11, Cell Signaling), GFP (1:1000; 598, MBL) and horseradish peroxidase-conjugated IgGs (1:1000; 7076 and 7074, HRP-linked Antibody, Cell Signaling; 1:1000; 18–8816-31 and 18–8817-33, TrueBlot, Rockland).

Techniques: Expressing

Journal: Mediators of Inflammation

Article Title: Bone Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Articular Cartilage Through Regulating tRF-Gln-TTG-019/UBL3

doi: 10.1155/mi/2705953

Figure Lengend Snippet: tRF-Gln-TTG-019 is regulated by BMSC-derived EVs to protect chondrocytes apoptosis and inflammation. (A) RNA was extracted from EVs derived from control or post-differentiated BMSCs. The differentially expressed tRFs were determined by high throughput sequencing. (B) The differentially expressed tRFs were validated by real-time PCR. Primary rat chondrocytes were treated by OGD/R and co-cultured with EVs derived from BMSCs and transfected with NC (OGD/R+EVs) or tRF-Gln-TTG-019 inhibitor (OGD/R+EVs+inhibitor), n = 5. After 48 h, cells were collected for PKH26 staining (C). The expression of tRF-Gln-TTG-019 was determined by real-time PCR, n = 5 (D). The cell proliferation was tested by CCK8 (E) and EdU staining (F). The cell apoptosis was determined by Annexin V/PI double staining (G) and TUNEL (H) assays. The content of IL-1b, IL-18, and LDH were detected by ELISA (I). The expression of NLRP3 and Caspase-1 was examined by Western blot (J). (K) Total cell lysates were immunoprecipitated with anti-UBL3 antibody and subsequently immunoblotted with anti-ubiquitin antibody. The expression of NLRP3 was detected by Western blot. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001. BMSC, bone mesenchymal stromal cell; EVs, extracellular vesicles; tRF, tRNA-derived fragment.

Article Snippet: Then, the membrane containing the cells was incubated overnight at 4°C with the following antibodies: anti-NLRP3 antibody (Abcam, Cambridge, MA, USA; 1:1000 diluted), anti-pro-caspase-1 antibody (Abcam; 1:1000 diluted), anti-cleaved-caspase-1 antibody (Abcam; 1:1000 diluted), anti-MMP3 (Abcam; 1:2000 diluted), anti-MMP13 (Abcam; 1:4000 diluted) or anti-UBL3 antibody (GeneTex; 1:1000 diluted).

Techniques: Derivative Assay, Control, Next-Generation Sequencing, Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Staining, Expressing, Double Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Mediators of Inflammation

Article Title: Bone Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Articular Cartilage Through Regulating tRF-Gln-TTG-019/UBL3

doi: 10.1155/mi/2705953

Figure Lengend Snippet: tRF-Gln-TTG-019 protects chondrocytes apoptosis and inflammation by targeting UBL3. (A) The predicated binding sites between tRF-Gln-TTG-019 and UBL3 untranslated region were shown. (B) Chondrocytes were co-transfected with UBL3 (wild type) and tRF-Gln-TTG-019 or mutant form of UBL3 and tRF-Gln-TTG-019. Cells were collected after transfection of 48 h. The luciferase activity was shown. (C, D) The mRNA and protein expression of UBL3 was determined by real-time PCR and Western blot. ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001. ns, no significant difference; tRF, tRNA-derived fragment.

Article Snippet: Then, the membrane containing the cells was incubated overnight at 4°C with the following antibodies: anti-NLRP3 antibody (Abcam, Cambridge, MA, USA; 1:1000 diluted), anti-pro-caspase-1 antibody (Abcam; 1:1000 diluted), anti-cleaved-caspase-1 antibody (Abcam; 1:1000 diluted), anti-MMP3 (Abcam; 1:2000 diluted), anti-MMP13 (Abcam; 1:4000 diluted) or anti-UBL3 antibody (GeneTex; 1:1000 diluted).

Techniques: Binding Assay, Transfection, Mutagenesis, Luciferase, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

Journal: Mediators of Inflammation

Article Title: Bone Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Articular Cartilage Through Regulating tRF-Gln-TTG-019/UBL3

doi: 10.1155/mi/2705953

Figure Lengend Snippet: Knockdown UBL3 suppresses the modulated function of tRF-Gln-TTG-019. Primary rat chondrocytes were treated with OGD/R, 4 h later, cells were transfected with NC or shUBL3 or co-transfected with shUBL3 and NC-EVs or inhibitor of tRF-Gln-TTG-019. The expression of UBL3 was determined by real-time PCR, n = 5 (A). The cell proliferation was tested by CCK8 (B) and EdU staining (C). The cell apoptosis was determined by Annexin V (D) and TUNEL (E) assays. The content of IL-1b, IL-18, and LDH were detected by ELISA (F). The expression of NLRP3, Caspase-1, MMP3, and MMP13 was examined by Western blot (G). (H) His-NLRP3 was overexpressed in chondrocytes. Total cell lysates were immunoprecipitated with anti-UBL3 antibody and subsequently immunoblotted with anti-ubiquitin antibody. The expression of His-NLRP3 was detected by Western blot. (I) Sulfated glycosaminoglycan (sGAGs) released from the culture medium were detected by ELISA. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001. tRF, tRNA-derived fragment.

Article Snippet: Then, the membrane containing the cells was incubated overnight at 4°C with the following antibodies: anti-NLRP3 antibody (Abcam, Cambridge, MA, USA; 1:1000 diluted), anti-pro-caspase-1 antibody (Abcam; 1:1000 diluted), anti-cleaved-caspase-1 antibody (Abcam; 1:1000 diluted), anti-MMP3 (Abcam; 1:2000 diluted), anti-MMP13 (Abcam; 1:4000 diluted) or anti-UBL3 antibody (GeneTex; 1:1000 diluted).

Techniques: Knockdown, Transfection, Expressing, Real-time Polymerase Chain Reaction, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Derivative Assay

Journal: Mediators of Inflammation

Article Title: Bone Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Articular Cartilage Through Regulating tRF-Gln-TTG-019/UBL3

doi: 10.1155/mi/2705953

Figure Lengend Snippet: UBL3 shRNA or BMSC-derived EVs ameliorate the defects of articular cartilage in rat knee joints. A model of defects in articular cartilage was established by a round full-thickness cartilage defects with a depth of 2.5 mm. The knee joint cartilage was collected and stained by H&E (A), toluidine blue (B), and TUNEL (C). Immunohistochemistry was used to detect Ki67 (D) and NLRP3 (E) (200× magnification). The synovial fluid was separated and subjected to ELISA assay (F). The protein levels of UBL3, NLRP3, and Caspase-1 in synovial fluid were tested by Western blot (G). (1) Sham, (2) CI, (3) CI+shNC, (4) CI+shUBL3, (5) CI+shUBL3+NC-EVs, (6) CI+shUBL3+inhibitor-EVs. ⁣ ∗∗∗ p < 0.001, n = 5. BMSC, bone mesenchymal stromal cell; EVs, extracellular vesicles; H&E, hematoxylin and eosin.

Article Snippet: Then, the membrane containing the cells was incubated overnight at 4°C with the following antibodies: anti-NLRP3 antibody (Abcam, Cambridge, MA, USA; 1:1000 diluted), anti-pro-caspase-1 antibody (Abcam; 1:1000 diluted), anti-cleaved-caspase-1 antibody (Abcam; 1:1000 diluted), anti-MMP3 (Abcam; 1:2000 diluted), anti-MMP13 (Abcam; 1:4000 diluted) or anti-UBL3 antibody (GeneTex; 1:1000 diluted).

Techniques: shRNA, Derivative Assay, Staining, TUNEL Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: UBL3 Interacts with Alpha-synuclein in Cells and the Interaction is Downregulated by the EGFR Pathway Inhibitor Osimertinib

doi: 10.1101/2023.05.15.540732

Figure Lengend Snippet: (A) Representative images of immunocytochemistry staining of p-S-129 α-syn in the sub-stantia nigra of WT and Ubl3 -/- mice. Scale bars: 10 μm. (B) Quantification comparison of the im-munohistochemical expression of p-S-129 α-syn in the substantia nigra between WT and Ubl3 -/- mice. Histograms represent the mean + SD (the number of mice in each group was three). *: p-value < 0.05. WT: wild type. Ubl3 -/- : Ubl3 knock out.

Article Snippet: Anti-p-S-129 α-syn antibody (Abcam, ab59264, 1:1000 dilution), anti-α-syn antibody (BioLegend, 834304, 1:1000 dilution), Anti-UBL3 antibody (ABclonal, A4028, 1:1000 dilution), anti-MYC antibody (MBL, M1932, 1:1000 dilution), anti-Flag antibody (MERCK, F7425-.2MG, 1:1000 dilution), horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell signaling, 7074, 1:5000 dilution), biotinylated anti-rabbit immunoglobulin G (Vector Laboratories, BA-1000, 1:1000 dilution).

Techniques: Immunocytochemistry, Staining, Expressing, Knock-Out

Journal: bioRxiv

Article Title: UBL3 Interacts with Alpha-synuclein in Cells and the Interaction is Downregulated by the EGFR Pathway Inhibitor Osimertinib

doi: 10.1101/2023.05.15.540732

Figure Lengend Snippet: (A) Schematic representation of the Gaussia princeps based split luciferase complementation assay system. (B) Schematic of split Gluc-tagged proteins, NGluc-UBL3, NGluc-UBL3Δ5, and α-syn-CGluc, both containing an IKSS sequence, which allows successfully expressed fragments and their interacting complexes to be secreted into the CM. (C) Immunoblotting of CM and CL from transfected HEK293 cells by anti-UBL3 polyclonal antibody and anti-α-syn antibody. (D) Luminescence of the CM (left) and CL (right) from transfected HEK293 cells. The luminescence ± SD in triplicate experiments is shown. (E) Schematic represen-tation of 3xFlag-UBL3, 3xFlag-UBL3Δ5, and 6xMYC-α-syn for co-immunoprecipitation (Co-IP). (F) Co-immunoprecipitated 3xFlag-UBL3 and 3xFlag-UBL3Δ5 interact with 6xMYC-α-syn. The input lanes are 1% of the sample prior to Co-IP, and the Co-IP lanes are 20% of the Co-IP products. IKSS: immunoglobulin kappa secretory signal; CM: cell culture medium; CL: cell lysate.

Article Snippet: Anti-p-S-129 α-syn antibody (Abcam, ab59264, 1:1000 dilution), anti-α-syn antibody (BioLegend, 834304, 1:1000 dilution), Anti-UBL3 antibody (ABclonal, A4028, 1:1000 dilution), anti-MYC antibody (MBL, M1932, 1:1000 dilution), anti-Flag antibody (MERCK, F7425-.2MG, 1:1000 dilution), horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Cell signaling, 7074, 1:5000 dilution), biotinylated anti-rabbit immunoglobulin G (Vector Laboratories, BA-1000, 1:1000 dilution).

Techniques: Luciferase, Sequencing, Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Cell Culture

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Schematic of CRISPR/Cas9 genetic screen. MutuDCs were lentivirally transduced with the GeCKOv2 library A and puromycin-resistant cells were stained for surface MHC II. The 5% highest MHC II expressing cells were sorted by flow cytometry, genomic DNA extracted, and amplified. gRNAs were quantified by Illumina NextSeq sequencing and analyzed with edgeR. Volcano plots show detected gRNAs enriched in sorted cells with increased MHC II expression compared to the unsorted library. P values were assessed using a negative binomial generalized linear model with a two-sided likelihood ratio test, with adjustment for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method. Guide RNAs with an absolute log2 fold change >1 and FDR of <0.01 were considered significantly enriched (orange dots), with genes hit by at least two gRNAs highlighted (large red circles). b Surface expression of MHC II, CD86, or MHC I were analyzed by flow cytometry for MutuDCs lacking B2m (pool), Marchf1, or Ubl3 (single-cell clones), compared with control cells expressing non-targeting hBim gRNA. Top: representative histograms, with gray solid histograms: control MutuDCs expressing non-targeting hBim gRNA, black lined histograms: CRISPR/Cas9 modified MutuDC lines as indicated. Bottom: quantification of flow cytometry analysis from three experiments. Bars indicate mean+SD of geometric mean fluorescence intensity (gMFI), relative to untransfected cells. Statistical analysis was performed using a one-way ANOVA and Bonferroni’s multiple comparisons test, comparing each sample to hBim controls, p values shown above bars, ns (not significant) p > 0.05.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: CRISPR, Transduction, Staining, Expressing, Flow Cytometry, Amplification, Sequencing, Clone Assay, Control, Modification, Fluorescence

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: Enriched gRNAs in the MHC II high population.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Analysis of Ii degradation and MHC II peptide loading in control MutuDCs expressing non-targeting hBim gRNA or Δ Ubl3 MutuDCs. Cells were metabolically labeled for 30 min, washed, and cultured in a growth medium for the indicated time points before lysis. MHC II molecules were immunoprecipitated with JV1 rabbit antisera, or normal rabbit serum (NRS), and protein G-Sepharose. Samples were loaded before (NB) or after boiling (B) at 95 °C ( b ), subjected to denaturing SDS-PAGE, transferred onto PVDF membranes, and exposed to a storage phosphor screen. Sizes corresponding to free MHC II α and β, full-length invariant chain (Ii), the Ii spliced variant Ii p41 , the degradation intermediate Ii p10 , and SDS-stable complexes αβIi p10 , and αβ-peptide (pep), are indicated on the right. Data from one experiment. b Δ Ubl3 MutuDCs or Δ Ubl3 MutuDCs expressing Myc-UBL3 were lysed and post-nuclear supernatants (PNS) were subjected to immunoprecipiation (IP) with anti-Myc-Tag agarose (clone 9E10) or anti-MHC II antibody (M5/114) crosslinked to protein G-sepharose. Per lane, samples equivalent to 5 × 10 6 cells (Myc IP), 2.5 × 10 6 cells (MHC II IP), or 400,000 cells (PNS) were analyzed by non-denaturing SDS-PAGE and western blotting using antibodies against UBL3 (ab113820), MHC II β chain (JV2), Myc (71D10), and ubiquitin (P4D1). Shown are representative blots and quantification of relative ubiquitination, with bars displaying mean + 95% confidence intervals for four experiments.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Control, Expressing, Metabolic Labelling, Labeling, Cell Culture, Lysis, Immunoprecipitation, SDS Page, Variant Assay, Western Blot, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a – d Clonal Δ Ubl3 MutuDCs were retrovirally transduced with Myc-tagged wild type UBL3 (“+Myc-UBL3 WT ”), or UBL3 C114S mutant (“+Myc-UBL3 C114S ”). a Post-nuclear supernatants of 2.5 × 10 5 cells were analyzed by non-denaturing SDS-PAGE and western blotting using anti-actin (20-33) anti-Myc (71D10) and anti-Ubl3 (ab113820) antibodies. Representative blot is shown for two independent experiments with similar results. b Proximity ligation assay (PLA). Δ Ubl3 MutuDC expressing Myc-UBL3 WT or Myc-UBL3 C114S were stained with plasma membrane (PM) CytoPainter. After washing, fixing, and permeabilization, cells were stained with Hoechst 33342, rabbit anti-MHC II (JV2) antiserum, and mouse anti-Myc mAb (9E10) and subjected to PLA. Scale bar = 10 µm. PLA spots indicate spatial proximity between MHC II and UBL3 and were enumerated within plasma membrane boundaries (violin plots). One experiment, P value as indicated, unpaired Welch’s t test (two-sided). c Immunofluorescence microscopy. Cells were fixed, permeabilized, and stained with biotinylated rat anti-MHC II mAb (M5/114), mouse anti-Myc mAb (9E10), streptavidin Alexa Fluor 647, donkey anti-mouse Alexa Fluor 594, and 0.5 μg/ml DAPI. Scale bar = 10 µm. Representative image shown for two independent experiments with similar results. d Flow cytometry analysis of MHC II and CD86. Left: Dot plots show MHC II ( x axis) and GFP ( y axis). Center: histograms show corresponding expression levels of MHC II or CD86, with gray solid histogram: control MutuDCs expressing non-targeting hBim gRNA; black line: Δ Ubl3 MutuDCs, red line: Δ Ubl3 MutuDCs expressing Myc-UBL3 WT , blue line: Δ Ubl3 MutuDCs expressing Myc-UBL3 C114S . Right: quantification of gMFI from three experiments, normalized to highest gMFI of each experiment, with bars mean ± SD and symbols. **** P < 0.0001, one-way ANOVA with Bonferroni’s test.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Transduction, Mutagenesis, SDS Page, Western Blot, Proximity Ligation Assay, Expressing, Staining, Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Ubl3 −/− mice. b Spleen cDC surface MHC II and CD86 by flow cytometry. Representative histograms and graphs showing gMFI relative to highest signal, bars mean ± SD, symbols individual mice ( n = 9, three independent experiments). One-way ANOVA with Bonferroni’s test. c Quantitative real-time PCR of H2-Ab1 and Marchf1 mRNA in spleen cDCs. Bars mean ± SD relative to WT, n = 2 samples, two experiments. d Spleen cDCs labeled with FIP-conjugated mAb for MHC II, CD86, or MHC I were incubated for 30 mins at 37°C. Fluorescence was quenched (+Q) and internalization was calculated as described in Methods. CD86 internalization not shown for cDC2 (low expression). Data representative of two independent experiments with similar results (MHC II) or pooled from two (CD86) or three (MHC I) experiments performed in triplicate, mean ± SD, one-way ANOVA with Bonferroni’s test. e MHC II was immunoprecipitated (IP) from spleen cDCs using anti-MHC II antibody, analyzed by non-denaturing SDS-PAGE, and immunoblotted (IB) for ubiquitin and MHC II β-chain. Left: representative blot. Right: MHC II ubiquitination relative to WT, bars mean + 95% confidence interval for three biological replicates. f – i Flow cytometry of surface MHC II and CD86 of f professional antigen-presenting cells (thymus: n = 14, four experiments (MHC II), n = 5, one experiment (CD86); blood: n = 7 for WT, 10 for Ubl3 −/− , 6 for Marchf1 −/− , two experiments; peritoneal cavity: n = 5 for WT, 4 for Ubl3 −/− , one experiment), g spleen myeloid cells ( n = 5 for WT, 4 for Ubl3 −/− , one experiment), h TEC ( n = 11 for WT, 12 for Ubl3 −/− , 12 for Marchf8 −/− , four experiments), and i lung epithelial cells ( n = 5, one experiment). Graphs show gMFI relative to highest gMFI, bars mean ± SD, symbols represent individual mice. One-way ANOVA with Bonferroni’s test, unpaired t test (two-sided) with Holm–Sidak correction. SPM small peritoneal macrophages, LPM large peritoneal macrophages, Mφ macrophages, mo monocytes, AECII type II lung alveolar epithelial cells, BEC bronchial epithelial cells, EC lung endothelial cells. p values above bars, with **** p < 0.0001, ns (not significant) p > 0.05.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Labeling, Incubation, Fluorescence, Expressing, Immunoprecipitation, SDS Page, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Left: spleen cDC1 expression of CLEC9A from wild type (WT) or Ubl3 −/− mice by flow cytometry, n = 11 mice examined over three experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). Right: Spleen cDCs from WT or Ubl3 −/− mice were labeled with FIP-conjugated anti-CLEC9A mAb. After 30 mins at 37°C, quencher (Q) was added and percentage internalization was calculated as described in Methods. Histograms show representative CLEC9A-FIP signal. Right: quantification of internalization from one experiment performed in triplicate, with bars mean ± SD, ns (not significant) p > 0.05, unpaired t test (two-sided). b In vivo antigen presentation assay. Purified CellTrace Violet (CTV)-labeled OT-I and OT-II cells were adoptively transferred into WT and Ubl3 −/− mice. 24 hours later, mice were injected with 0.2 µg anti-CLEC9A mAb-targeted OVA and spleens harvested after 64 hours. Antigen presentation capacity was assessed by flow cytometric analysis of CTV-dilution by OT-I and OT-II proliferation. Data from n = 9 (OT-I) and n = 8 for WT, 7 for Ubl3 −/− (OT-II) mice, two experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). c Quantification of spleen cDC1 and cDC2 from WT or Ubl3 −/− mice. Data from n = 9 mice, three independent experiments, bars mean ± SD, symbols representing individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). d Left: C3 expression on spleen cDCs from WT, Ubl3 −/− or Marchf1 −/− mice by flow cytometry. Data from n = 5 from one experiment, bars mean ± SD, symbols represent individual mice. Two-way ANOVA with Bonferroni’s test. Right: Quantification of trogocytic B cells gated on B220 + CD19 + cells in Nycodenz-enriched splenocytes from WT, Ubl3 −/− or Marchf1 −/− mice. Representative dot plots and data from one experiment with n = 5, bars mean ± SD, and symbols representing individual mice. Significant p values shown above bars, with **** p < 0.0001, ns (not significant) p > 0.05, one-way ANOVA with Bonferroni’s test.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Expressing, Flow Cytometry, Labeling, In Vivo, Immunopeptidomics, Purification, Injection

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Ex vivo antigen presentation assay. WT and Ubl3 −/− mice were injected with 1 µg of anti-CLEC9A-OVA mAb. Indicated numbers of spleen cDC1 and cDC2 were purified and co-cultured with OT-I or OT-II cells, and divided OT-I or OT-II cells enumerated by flow cytometry. Data with n = 3, representative of two experiments, mean ± SD, **** p < 0.0001, two-way ANOVA with Bonferroni’s test. b Flow cytometry analysis of relative cell surface marker expression of spleen cDC1 or cDC2 isolated from WT and Ubl3 −/− mice, with n = 8 mice, two independent experiments, symbols represent individual mice, bars mean ± SD, **** p < 0.0001, unpaired t test (two-sided) with Holm–Sidak adjustment. c OVA-Cy5 uptake assay. Purified spleen cDC1 or cDC2 from WT or Ubl3 −/− mice were incubated with 50 µg/ml OVA-Cy5 for the indicated times, and washed before flow cytometry analysis. Graphs show mean ± SD, with data from one experiment performed in triplicate, two-way ANOVA with Bonferroni’s test. d Proteolysis assay. Purified spleen cDC1 or cDC2 from WT or Ubl3 −/− mice were pulsed with DQ-OVA for 15 min, washed twice and DQ-OVA signal was measured by flow cytometry at different chase time points. Graphs show mean ± SD, with data pooled from two independent experiments performed in triplicate, two-way ANOVA with Bonferroni’s test. e Quantification of cathepsin (cat) activity in spleen cDCs. Top: representative gel indicating active cathepsin X/B/S/L, and actin immunoblot. Bottom: relative protease activity normalized to actin, with bars showing mean ± SEM of one experiment with three biological replicates. Four spleens were pooled for each biological replicate, ns not significant, unpaired t test (two-sided). f Cytokine expression of purified Ubl3 −/− and wild-type spleen cDC1 and cDC2 stimulated with CpG, IFN-γ, and GM-CSF. Bars display mean ± SD of cells stimulated in duplicate, using purified cDCs pooled from eight mice, representative of two independent experiments.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Ex Vivo, Immunopeptidomics, Injection, Purification, Cell Culture, Flow Cytometry, Marker, Expressing, Isolation, Incubation, Proteolysis Assay, Activity Assay, Western Blot

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: Human CD14 + monocytes were infected with non-targeting control shRNA or shRNA against UBL3 (sh1 UBL3 or sh2 UBL3 ) and analyzed after five days in culture. Top left: Cell lysates were probed with antibodies against UBL3 (LS-C661402) and actin, and quantification of gene silencing was determined by UBL3 signal intensity. Immunoblot depicts a scenario of approximately 95% UBL3 knockdown. Data from n = 6 donor samples examined in a single experiment, with symbols connected with a line representing individual donors. Top right: Surface MHC II, CD86, and MHC I on monocyte-derived DCs (CD16 − CD1a + ) and monocyte-derived macrophages (CD16 + CD1a − ) were analyzed by flow cytometry. Histograms are representative of analysis from one donor. Bottom: Graphs showing data from n = 6 (DCs) or n = 4 (macrophages) donor samples examined in a single experiment, with symbols connected with a line representing individual donors, p values shown above points, ns not significant, repeated measures one-way ANOVA.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Infection, Control, shRNA, Western Blot, Knockdown, Derivative Assay, Flow Cytometry