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$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 <t>CD63-iRFP670</t> (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.$
Irfp670, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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irfp670 - by Bioz Stars, 2026-05

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1) Product Images from "Intracellular dynamics of ubiquitin-like 3 visualized using an inducible fluorescent timer expression system"

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

Journal: Biology Open

doi: 10.1242/bio.060345

$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.$
Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Marker

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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.

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.

Techniques: Marker

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/eLife.76651

Figure Lengend Snippet:

Article Snippet: Lentiviral pCDH-antiGFPnanobody-mCherry-Rab11a (overexpressing control) was cloned using pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a (previously prepared in our lab) via SpeI/SalI and XbaI/XhoI complementary cleavage sites. pMito-mCherry-FRB , a gift from Stephen Royle (Addgene plasmid #59352), was used as a template for generation of pMito-iRFP670-FRB (used with GFP-FKBP for knocksideways). pSH-EFIRES-P-AtAFB2 (Addgene plasmid #129715) , pCDH-EF1-tagBFP-T2A-mycBirA*-Rab11a, and mTagBFP2 were used to generate lentiviral pCDH-AtAFB2-mTagBFP2.

Techniques: Modification, Transfection, Construct, shRNA, Recombinant, Expressing, Plasmid Preparation, CRISPR, Knock-In, Labeling, Sequencing, Software, Invasion Assay

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