ubl3  (Proteintech)

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: Biology Open

Article Title: Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system

doi: 10.1242/bio.060345

Figure Lengend Snippet: Design and construction of a system for spatiotemporal visualization of UBL3 localization. (A) Schematic diagram of plasmids and the Tet-On inducible system. The pCAG-Tet-on 3G is a regulatory plasmid containing DOX-inducible reverse tetracycline transactivator (rtTA) under the cytomegalovirus (CMV) promoter. The pTRE tight -medium-FT-UBL3 is a response plasmid containing the expression cassette of UBL3 fused to the C-terminus of the medium-fluorescent timer (FT) under the promoter comprising tetracycline response element (TRE) and minimal cytomegalovirus promoter (CMV mini). The cells were transfected with pCAG-Tet-on 3G plasmid and pTRE tight -medium-FT-UBL3 plasmid. When DOX was added to the medium, it bound to rtTA, which in turn was bound to TRE and induces medium-FT-UBL3 gene expression. When DOX was washed out, rtTA was dissociated from TRE, and gene expression was terminated. (B) Representative sum intensity projection sequential images of identical MDA-MB-231 cells transfected with FT-UBL3 after 4, 8, 12, and 24 h of DOX treatment. Green pseudo-color, blue form of FT-UBL3. Magenta pseudo-color, red form of FT-UBL3. Scale bar: 50 µm. (C,D) Time changes of the blue (green line and circles) and red (magenta line and circles) sum fluorescence intensities (C) and ratio value (red form/blue form) (D) of FT-UBL3-expressing cells. The orange line indicates DOX induction time window. Data are presented as mean±s.e.m. Light colored lines depict data from individual cell ( n =16 cells from three independent experiments).

Article Snippet: Anti-UBL3 (14100-1-AP, Proteintech, 1:1000), anti-Vinculin (EPR8185, Abcam, 1:1000), anti-Carleticulin (#2891, Cell Signaling Technology, 1:500), anti-Caveolin 1 (610407, BD Biosciences, 1:500) and HRP-conjugated secondary antibody (anti-rabbit IgG, #7074S, Cell Signaling Technology, 1:1000; anti-mouse IgG, #7076S, Cell Signaling Technology, 1:1000) were used in the experiments.

Techniques: Plasmid Preparation, Expressing, Transfection, Fluorescence

Journal: Biology Open

Article Title: Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system

doi: 10.1242/bio.060345

Figure Lengend Snippet: The subcellular route of UBL3 transported to multivesicular bodies. (A) Representative maximum intensity projection images of different MDA-MB-231 cells transfected with FT-UBL3 plus CD63-iRFP670 (a multivesicular body marker) after 4, 8, 14, 20, and 24 h of DOX treatment. Green pseudo-color, blue form of FT-UBL3. Magenta pseudo-color, red form of FT-UBL3. Scale bar: 20 µm. (B) Quantitative analysis of the blue (after 4, 6, 8, and 10 h of DOX treatment) and red (after 12, 14, 16, 18, 20, 22, and 24 h of DOX treatment) fluorescence intensities of FT-UBL3-expressing cells in CD63-positive MVBs. (C) Representative vertical cross-sections of different MDA-MB-231 cells transfected with FT-UBL3 and labeling of the plasma membrane using the CellMask Deep Red plasma membrane stain. Yellow squares indicate cropped areas. The white dashed lines indicate the boundary between the cytosol and the plasma membrane defined by the CellMask plasma membrane stain. Arrowheads indicate the localization of FT-UBL3. Scale bars: 5 µm and 0.3 µm. (D) Quantitative analysis of blue (after 4, 6, 8, and 10 h of DOX treatment) and red (after 12, 14, 16, 18, 20, 22, and 24 h of DOX treatment) fluorescence intensities of FT-UBL3-expressing cells in the CellMask-positive basal area. (E) Western blot analysis of cytosolic and membrane fractions of FT-UBL3 expressing MDA-MB-231 cells after 4, 8, and 24 h of DOX treatment using antibodies against Vinculin (cytosolic marker), Calreticulin (organelle membrane marker) and Caveolin 1 (plasma membrane marker). (F) Quantitative analysis of the ratio of FT-UBL3 protein in the cytosolic fraction relative to the membrane fraction, with 0.017 μg of protein. Data are presented as mean±s.e.m. Dots indicate data from individual cells ( n =17–28 cells from more than three independent experiments) (B,D) or individual experiments ( n =5) (F). One-way analysis of variance with Tukey's multiple comparison test. P -values are shown at the top of the graphs only for the time periods in which significant differences were found for B, D and F. *** P <0.001, ** P <0.01, * P <0.05.

Article Snippet: Anti-UBL3 (14100-1-AP, Proteintech, 1:1000), anti-Vinculin (EPR8185, Abcam, 1:1000), anti-Carleticulin (#2891, Cell Signaling Technology, 1:500), anti-Caveolin 1 (610407, BD Biosciences, 1:500) and HRP-conjugated secondary antibody (anti-rabbit IgG, #7074S, Cell Signaling Technology, 1:1000; anti-mouse IgG, #7076S, Cell Signaling Technology, 1:1000) were used in the experiments.

Techniques: Transfection, Marker, Fluorescence, Expressing, Labeling, Membrane, Staining, Western Blot, Comparison

Journal: Biology Open

Article Title: Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system

doi: 10.1242/bio.060345

Figure Lengend Snippet: Characterization of a system for spatiotemporal dynamics of UBL3 mutants, C113/114A (lacking both UBL3 modification activity and membrane localization) and UBL3Δ1 (retaining membrane localization, lacking UBL3 modification activity). (A,D) Representative sequential images of identical MDA-MB-231 cells transfected with FT-UBL3C113/114A (A) and FT-UBL3Δ1 (D) after 4, 8, 12, and 24 h of DOX treatment. Scale bars: 50 µm. (B,E) Time changes of the blue (green line and circles) and red (magenta line and circles) sum fluorescence intensities of FT-UBL3 C113/114A (B) and FT-UBL3Δ1 expressing cells (E). (C,F) Time changes in the ratio of red form/blue form of FT-UBL3 C113/114A (C) and FT-UBL3Δ1 expressing cells (F). The orange line indicates DOX induction time window. Data are presented as mean±s.e.m. Light colored lines depict data from individual cell ( n =12 cells from more than three independent experiments).

Article Snippet: Anti-UBL3 (14100-1-AP, Proteintech, 1:1000), anti-Vinculin (EPR8185, Abcam, 1:1000), anti-Carleticulin (#2891, Cell Signaling Technology, 1:500), anti-Caveolin 1 (610407, BD Biosciences, 1:500) and HRP-conjugated secondary antibody (anti-rabbit IgG, #7074S, Cell Signaling Technology, 1:1000; anti-mouse IgG, #7076S, Cell Signaling Technology, 1:1000) were used in the experiments.

Techniques: Modification, Activity Assay, Membrane, Transfection, Fluorescence, Expressing

Journal: Biology Open

Article Title: Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system

doi: 10.1242/bio.060345

Figure Lengend Snippet: The spatiotemporal localization of UBL3C113/114A (lacking both UBL3 modification activity and membrane localization) and UBL3Δ1 (retaining membrane localization, lacking UBL3 modification activity). (A,C) Representative maximum intensity projection images of different MDA-MB-231 cells transfected with FT-UBL3C113/114A (A) and FT-UBL3Δ1 (C) plus CD63-iRFP670 (a multivesicular body marker) after 4, 8, 14, and 24 h of DOX treatment. Green pseudo-color, blue form of FT-UBL3C113/114A and FT-UBL3Δ1. Magenta pseudo-color, red forms of FT-UBL3C113/114A and FT-UBL3Δ1. Arrowheads indicate the cell periphery. Scale bars: 20 µm. (B,D) Quantitative analysis of the blue (after 4, 6, 8, and 10 h of DOX treatment) and red (after 12, 14, 16, 18, 20, 22, and 24 h of DOX treatment) fluorescence intensities of FT-UBL3C113/114A and FT-UBL3Δ1-expressing cells in CD63-positive MVBs. (E) Representative vertical cross-sections of different MDA-MB-231 cells transfected with FT-UBL3Δ1 and labeling of the plasma membrane using the CellMask Deep Red plasma membrane stain. Yellow squares indicate cropped areas. The white dashed lines indicate the boundary between the cytosol and the plasma membrane defined by the CellMask plasma membrane stain. Arrowheads indicate the localization of FT-UBL3Δ1. Scale bars: 5 µm and 0.3 µm. (F) Quantitative analysis of the blue (after 4, 6, 8, and 10 h of DOX treatment) and red (after 12, 14, 16, 18, 20, 22, and 24 h of DOX treatment) fluorescence intensities of FT-UBL3Δ1-expressing cells in the CellMask-positive plasma membrane. Data are presented as mean±s.e.m. Dots indicate data from individual cells ( n =12–20 cells from more than three independent experiments). One-way analysis of variance with Tukey's multiple comparison test. P -values are shown at the top of the graphs only for the time periods in which significant differences were found for B, C, D, F, G, and H. *** P <0.001, ** P <0.01, and * P <0.05.

Article Snippet: Anti-UBL3 (14100-1-AP, Proteintech, 1:1000), anti-Vinculin (EPR8185, Abcam, 1:1000), anti-Carleticulin (#2891, Cell Signaling Technology, 1:500), anti-Caveolin 1 (610407, BD Biosciences, 1:500) and HRP-conjugated secondary antibody (anti-rabbit IgG, #7074S, Cell Signaling Technology, 1:1000; anti-mouse IgG, #7076S, Cell Signaling Technology, 1:1000) were used in the experiments.

Techniques: Modification, Activity Assay, Membrane, Transfection, Marker, Fluorescence, Expressing, Labeling, Staining, Comparison

Journal: Biology Open

Article Title: Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system

doi: 10.1242/bio.060345

Figure Lengend Snippet: Association of UBL3 and α-tubulin, its substrate, in the cytosol and transport to MVBs. (A) Representative super-resolution images show bright spots of α-tubulin-iRFP670 with a long diameter of 200–800 nm in the cytosol, the blue form of FT-UBL3, and TagRFP-CD63 (a MVB marker). Arrowheads indicate bright spots of α-tubulin not colocalizing with FT-UBL3. Scale bar: 1 µm. (B) Quantitative analysis of the percentage of cells with bright spots of α-tubulin with a long diameter of 200–800 nm in the cytosol. (C) Representative super-resolution images show bright spots of α-tubulin-iRFP670 colocalized with the blue form of FT-UBL3 and TagRFP-CD63. Arrows indicate bright spots of α-tubulin colocalizing with FT-UBL3. Scale bar: 1 µm. (D) Quantitative analysis of the percentage of cells with bright spots of α-tubulin co-localized with FT-UBL3 in the cytosol divided by the number of cells that have bright spots of α-tubulin in the cytosol. (E) Representative super-resolution time-lapse images to track the bright spot of α-tubulin colocalized with the blue form of FT-UBL3 and TagRFP-CD63. Scale bar: 500 nm. Data are presented as min to max. Dots indicate data from independent experiments ( n =14 independent experiments). Kruskal–Wallis with Dunn's test. P -values are shown at the top of the graphs. *** P <0.001, ** P <0.01, and * P <0.05.

Article Snippet: Anti-UBL3 (14100-1-AP, Proteintech, 1:1000), anti-Vinculin (EPR8185, Abcam, 1:1000), anti-Carleticulin (#2891, Cell Signaling Technology, 1:500), anti-Caveolin 1 (610407, BD Biosciences, 1:500) and HRP-conjugated secondary antibody (anti-rabbit IgG, #7074S, Cell Signaling Technology, 1:1000; anti-mouse IgG, #7076S, Cell Signaling Technology, 1:1000) were used in the experiments.

Techniques: Marker