gfp cd63  (Addgene inc)

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A) Immunoblotting analysis of STX1A knockdown and control cells for checking the expression of STX1A. * non-specific band highlighted by anti-STX1A antibody. The fold change in band intensities was normalized with internal control and indicated on the blots. (B) Immunoblotting analysis of GFP-STX1A overexpression in HeLa cells. GFP expression is used as a control. The blot was probed with an anti-GFP antibody. γ-tubulin is used as the loading control in all immunoblots. (C) Images represent the GFP-STX1A overexpression in HeLa cells. Undeconvolved (left) and deconvolved (right) images are shown separately. Insets are magnified views of the white boxed areas. (D-F) Live cell imaging microscopy of HeLa cells expressing GFP-STX1A and LAMP1-RFP in D; mCherry-STX1A and GFP-CD63 in E; and GFP-CD63 and LAMP1-RFP in F. Insets are magnified views of the white boxed areas shown in the images at different time points (s, Sec). Scale bars, 10 µm. (G) Subcellular membrane fractionation of HeLa cells. Fractions were probed for STX1A, LAMP1 (lysosome marker), Na + -K + ATPase (plasma membrane marker) and Rab22A (recycling endosome marker). In- Input and numbers represent the membrane (alternative) fractions from top to bottom of the sucrose gradient. (H) Plot represents the number of LysoTracker puncta in non-transfected (-) and GFP-STX1A overexpressing cells. (I) Plot represents the CTCF (A.U.) of DQ-Red intensity in non-transfected (-) and GFP-STX1A overexpressing cells. (J) Plot represents the size of lysosomes (μm 2 ) in non-transfected (-) and GFP-STX1A overexpressing cells. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant and *p≤0.1.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Western Blot, Knockdown, Control, Expressing, Over Expression, Live Cell Imaging, Microscopy, Membrane, Fractionation, Marker, Transfection

Journal: bioRxiv

Article Title: STX1A localizes to the lysosome and controls its exocytosis

doi: 10.1101/2025.03.29.646068

Figure Lengend Snippet: (A, E) IFM analysis of HeLa cells transfected with control or two different STX1A (1 and 2) siRNA sequences. The cells were stained for LAMP1 and LBPA or CD63 (A). Another set of cells was overexpressed with RFP-LC3 or tfLC3 (rat LC3 fused to mRFP and EGFP, E). RFP-LC3 expressing cells were stained for LAMP1. Individual and merged panels are shown separately. Insets are magnified views of the white boxed areas. Scale bars, 10 µm. (B) Plot represents the Pearson’s correlation coefficient ( r ) between LAMP1 and LBPA, shown in A. (C, D, G) Immunoblotting analysis of control and STX1A knockdown cell lysates. The blots were probed for checking the expression of different lysosome associated proteins in C, cargo proteins in D, and autophagy related proteins in G. γ- tubulin is used as the loading control in all immunoblots. The fold change in band intensities was normalized with internal control and indicated on the blots. (F) Plots represent the number of RFP-LC3 puncta, Pearson’s correlation coefficient ( r value) between LAMP1 and RFP-LC3, and the number of autolysosomes. The average values in mean±s.e.m. are indicated on the graphs. N=3. ns-non-significant, **p≤0.01, and ***p≤0.001.

Article Snippet: Other plasmids: mCherry-UtrCh (26740), GFP-CD63 (62964), LAMP1-GFP (34831), LAMP1-RFP (1817), pMD2.G (VSV-G lentiviral envelop vector, 12259), psPAX2 (lentiviral packaging vector, 12260), pmRFP-LC3 (21075) and ptLC3 (rat LC3 fused to mRFP and EGFP, 21074) were obtained from Addgene.

Techniques: Transfection, Control, Staining, Expressing, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Hyaluronic acid hydrogels: Establishing a sustained delivery system for extracellular vesicles

doi: 10.1101/2025.01.29.635521

Figure Lengend Snippet: Characterization of EVs harvested from the bioreactor system (i) TEM imaging demonstrating characteristic lipid bi-layer (A) 120,000x (B) 200,000x (ii) Protein Concentrations of EVs obtained from early, intermediate and late EV harvests (Fractions 1-7) (iii) Western blot analysis of GFP-sEVs positive for tetraspanin CD63 (sEVs Sample 1), ESCRT pathway marker TSG 101 (sEVs sample S1 and S2 and cells; higher expression observed in cells) and negative for endoplasmic marker calnexin in (sEVs sample S3) and observed in cell samples (S1 and S2) (iv) GFP expression observed in EV uptake studies by confocal imaging (A) Control wild type MDA-MB231 cells stained with DAPI co cultured with no EVs (60X Magnification) (B) Wild type MDA-MB231 cells stained with DAPI co cultured with GFP EVs (60X Magnification), red arrows showing uptake of GFP EV clusters.

Article Snippet: MDA-MB-231 triple negative breast cancer cells (LGC Limited, UK) stably transduced with Exosome Cyto-Tracer, pCT-CD63-GFP (System Biosciences) were used for EV production.

Techniques: Imaging, Western Blot, Marker, Expressing, Control, Staining, Cell Culture

Journal: bioRxiv

Article Title: Harnessing Extracellular Vesicles for Stabilized and Functional IL-10 Delivery in Macrophage Immunomodulation

doi: 10.1101/2025.01.14.633016

Figure Lengend Snippet: a) Size distribution and concentration were measured using nanoparticle tracking analysis. b) Western blot analysis of IL-10 protein showing multiple bands and EV markers CD9, CD63, and CD81. c) IL-10 mRNA expression analyzed by qPCR. d) Proteinase K protection assay for IL-10 + sEVs, with TSG101 serving as a luminal protein control. e) Stability comparison between sEV-incorporated IL-10 and recombinant human IL-10 protein (RP) under 2 h at room temperature (RT), 2 h at 37 °C (37), and 2 cycles of freezing and thawing (F/T). Fresh samples served as controls. Naïve sEVs were isolated from non-transfected HEK293FT cells.

Article Snippet: Human CD63-GFP (Addgene plasmid #62964, gift from Paul Luzio) was subcloned into the pKT2/CAGXSP vector using recombination cloning (In-Fusion HD Cloning Kit, Clontech), as described before [ , ].

Techniques: Concentration Assay, Western Blot, Expressing, Control, Comparison, Recombinant, Isolation, Transfection

Journal: bioRxiv

Article Title: Harnessing Extracellular Vesicles for Stabilized and Functional IL-10 Delivery in Macrophage Immunomodulation

doi: 10.1101/2025.01.14.633016

Figure Lengend Snippet: a) The isolated sEVs underwent a second purification step based on their surface charge by anion exchange chromatography with a linear NaCl gradient (30-500 mM) (Created with BioRender.com ). b) Representative transmission electron microscopy images of sEVs in Protein-high (left), Protein-low (middle), and Flow-through (right) fractions purified from IL-10 + sEVs. c) Western blot analysis of IL-10 protein and Exo markers CD9, CD63 and CD81 in Protein-high fractions 9-12. d) Western blot analysis comparing IL-10 protein expression between naïve and IL-10 + Exos. e) qPCR analysis of pro-inflammatory mRNA markers in LPS-stimulated THP-1 macrophages following 24 h of incubation with 10 µg of Exos.

Article Snippet: Human CD63-GFP (Addgene plasmid #62964, gift from Paul Luzio) was subcloned into the pKT2/CAGXSP vector using recombination cloning (In-Fusion HD Cloning Kit, Clontech), as described before [ , ].

Techniques: Isolation, Purification, Chromatography, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Incubation

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: CD63 from pCT-CD63-GFP (CYTO120-PA-1, System Biosciences) and TagRFP, or iRFP670 were subcloned into pcDNA3. pTubulin-iRFP670 was a gift from Kiryl Piatkevich (Addgene plasmid # 197237; http://n2t.net/addgene:197237 ; RRID:Addgene_197237).

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: 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: CD63 from pCT-CD63-GFP (CYTO120-PA-1, System Biosciences) and TagRFP, or iRFP670 were subcloned into pcDNA3. pTubulin-iRFP670 was a gift from Kiryl Piatkevich (Addgene plasmid # 197237; http://n2t.net/addgene:197237 ; RRID:Addgene_197237).

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: CD63 from pCT-CD63-GFP (CYTO120-PA-1, System Biosciences) and TagRFP, or iRFP670 were subcloned into pcDNA3. pTubulin-iRFP670 was a gift from Kiryl Piatkevich (Addgene plasmid # 197237; http://n2t.net/addgene:197237 ; RRID:Addgene_197237).

Techniques: Marker