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egfp rab18q67l  (Addgene inc)


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    Structured Review

    Addgene inc egfp rab18q67l
    GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells <t>expressing</t> <t>EGFP‐Rab18Q67L</t> (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.
    Egfp Rab18q67l, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling"

    Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

    Journal: Journal of Extracellular Biology

    doi: 10.1002/jex2.70112

    GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells expressing EGFP‐Rab18Q67L (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.
    Figure Legend Snippet: GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells expressing EGFP‐Rab18Q67L (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.

    Techniques Used: Microscopy, Stable Transfection, Expressing, Immunofluorescence, Staining



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    GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells <t>expressing</t> <t>EGFP‐Rab18Q67L</t> (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.
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    Hsp90α and nSMase2 are effectors of GDP‐bound <t>Rab18.</t> (A) GST pull‐down assays using mouse brain lysates incubated with GST‐tagged Rab18 mutants, immunoblotted with the indicated antibodies. Rab18WT and Rab18Q67L preferentially associated with kinesin‐1 (KIF5A), whereas Rab18S22N preferentially bound Hsp90α and nSMase2. (B) Schematic summary of Rab18‐GTP– and Rab18‐GDP–specific effector interactions. (C, D) Spinning disk microscopy of NIH3T3 cells co‐expressing Hsp90α‐tagRFP <t>and</t> <t>EGFP‐Rab18</t> mutants (C), with quantification of cells showing perinuclear Hsp90α accumulation (D). Open arrows, diffuse cytoplasmic distribution of Hsp90α in a Rab18‐GTP‐expressing cell. Scale bar, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. (E, F) Ceramide immunocytochemistry of NIH3T3 cells treated with DMSO, CID (40 µM), or CID plus GW4869 (1.25 µM, 24 h), shown at low and high magnification (E), with quantification (F). Arrows indicate peripheral ceramide accumulation. Scale bars, 20 µm. * p < 0.05; ** p < 0.01; ***p < 0.001; one‐way ANOVA; n = 20.
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    ( A,B ) Cytoplasmic GFP and mCherry-Mem <t>transfected</t> NIH3T3 cells were immuno-stained for GFP under ( A ) permeabilized or ( B ) non-permeabilized conditions. GFP is detected by anti-GFP in permeabilized cells, but not in non-permeabilized cells (asterisks). ( C,D ) SHH-GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained using anti-SHH under, ( C ) permeabilized or, ( D ) non-permeabilized conditions. SHH-GFP signal is evident in cytonemes in both conditions (yellow arrows), but not evident in non-permeabilized cells when probed with anti-SHH (red arrows). ( E–G ) Cytonemes of NIH3T3 cells expressing SHH-mCherry and CD63-EGFP ( E ) or Rab18-EGFP ( G ) analyzed by confocal microscopy do not show significant co-localization. Rab18-EGFP does not enter cytonemes. ( F ) Box plot with min/max whiskers of Pearson’s coefficients values of colocalization between SHH-mCherry and CD63-EGFP in cytonemes.
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    ( A,B ) Cytoplasmic GFP and mCherry-Mem <t>transfected</t> NIH3T3 cells were immuno-stained for GFP under ( A ) permeabilized or ( B ) non-permeabilized conditions. GFP is detected by anti-GFP in permeabilized cells, but not in non-permeabilized cells (asterisks). ( C,D ) SHH-GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained using anti-SHH under, ( C ) permeabilized or, ( D ) non-permeabilized conditions. SHH-GFP signal is evident in cytonemes in both conditions (yellow arrows), but not evident in non-permeabilized cells when probed with anti-SHH (red arrows). ( E–G ) Cytonemes of NIH3T3 cells expressing SHH-mCherry and CD63-EGFP ( E ) or Rab18-EGFP ( G ) analyzed by confocal microscopy do not show significant co-localization. Rab18-EGFP does not enter cytonemes. ( F ) Box plot with min/max whiskers of Pearson’s coefficients values of colocalization between SHH-mCherry and CD63-EGFP in cytonemes.
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    Image Search Results


    GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells expressing EGFP‐Rab18Q67L (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.

    Journal: Journal of Extracellular Biology

    Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

    doi: 10.1002/jex2.70112

    Figure Lengend Snippet: GDP‐bound Rab18 accumulates in perinuclear endosomes and promotes SHH‐XLEV secretion. (A–C) Airyscan microscopy of NIH3T3 cells stably expressing SHHN‐EGFP following treatment with ZSTK474 (1 µM, 1 h; A, upper), LY294002 (50 µM, 8 h; A, lower), or SF1670 (250 nM, overnight; B), with quantification of peripheral distribution scores (PDS; C). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 20. (D–F) Airyscan microscopy of NIH3T3 cells expressing EGFP‐Rab18Q67L (GTP‐bound mimetic; D) or EGFP‐Rab18S22N (GDP‐bound mimetic; E), with PDS quantification (F). Scale bars, 20 µm. ** p < 0.01; Welch's t test; n = 7–16. (G–I) Airyscan immunofluorescence microscopy of EGFP‐Rab18S22N–expressing cells stained for AP‐1 (G) or AP‐3 (H) at low (upper) and high (lower) magnifications, with colocalization analysis (I). Arrowheads indicate perinuclear Rab18S22N puncta. Scale bars, 20 µm. *** p < 0.001; Welch's t test; n = 10.

    Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

    Techniques: Microscopy, Stable Transfection, Expressing, Immunofluorescence, Staining

    Hsp90α and nSMase2 are effectors of GDP‐bound Rab18. (A) GST pull‐down assays using mouse brain lysates incubated with GST‐tagged Rab18 mutants, immunoblotted with the indicated antibodies. Rab18WT and Rab18Q67L preferentially associated with kinesin‐1 (KIF5A), whereas Rab18S22N preferentially bound Hsp90α and nSMase2. (B) Schematic summary of Rab18‐GTP– and Rab18‐GDP–specific effector interactions. (C, D) Spinning disk microscopy of NIH3T3 cells co‐expressing Hsp90α‐tagRFP and EGFP‐Rab18 mutants (C), with quantification of cells showing perinuclear Hsp90α accumulation (D). Open arrows, diffuse cytoplasmic distribution of Hsp90α in a Rab18‐GTP‐expressing cell. Scale bar, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. (E, F) Ceramide immunocytochemistry of NIH3T3 cells treated with DMSO, CID (40 µM), or CID plus GW4869 (1.25 µM, 24 h), shown at low and high magnification (E), with quantification (F). Arrows indicate peripheral ceramide accumulation. Scale bars, 20 µm. * p < 0.05; ** p < 0.01; ***p < 0.001; one‐way ANOVA; n = 20.

    Journal: Journal of Extracellular Biology

    Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

    doi: 10.1002/jex2.70112

    Figure Lengend Snippet: Hsp90α and nSMase2 are effectors of GDP‐bound Rab18. (A) GST pull‐down assays using mouse brain lysates incubated with GST‐tagged Rab18 mutants, immunoblotted with the indicated antibodies. Rab18WT and Rab18Q67L preferentially associated with kinesin‐1 (KIF5A), whereas Rab18S22N preferentially bound Hsp90α and nSMase2. (B) Schematic summary of Rab18‐GTP– and Rab18‐GDP–specific effector interactions. (C, D) Spinning disk microscopy of NIH3T3 cells co‐expressing Hsp90α‐tagRFP and EGFP‐Rab18 mutants (C), with quantification of cells showing perinuclear Hsp90α accumulation (D). Open arrows, diffuse cytoplasmic distribution of Hsp90α in a Rab18‐GTP‐expressing cell. Scale bar, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. (E, F) Ceramide immunocytochemistry of NIH3T3 cells treated with DMSO, CID (40 µM), or CID plus GW4869 (1.25 µM, 24 h), shown at low and high magnification (E), with quantification (F). Arrows indicate peripheral ceramide accumulation. Scale bars, 20 µm. * p < 0.05; ** p < 0.01; ***p < 0.001; one‐way ANOVA; n = 20.

    Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

    Techniques: Incubation, Microscopy, Expressing, Immunocytochemistry

    GDP‐bound Rab18 promotes perinuclear SHH‐XLEV secretion. (A–C) Spinning disk microscopy of NIH3T3 cells coexpressing SHHN‐tagRFP and EGFP‐Rab18 mutants, shown as z‐projections (A), single planes (A), and side views (B), with quantification of perinuclear SHH accumulation (C). Dotted lines, periphery of the Rab18‐GTP‐expressing colony (green) and basolateral SHH deposition (magenta). Arrowheads, colocalization spots of Rab18‐GTP and SHH on the plasma membrane (A) and Rab18‐GTP‐specific basolateral SHH deposition (B). Scale bars, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. Related to . (D) Spinning disk microscopy of NIH3T3 cells undergoing burst‐like secretion of SHHN‐EGFP and Hsp90α‐tagRFP following prolonged CID treatment (arrowheads, 30 h). Scale bar, 20 µm. Related to Figure and . (E, F) Three‐dimensional reconstruction of surface SHH immunofluorescence (red) in SHHN‐EGFP–expressing NIH3T3 cells (E), with quantification of SHH signal intensity appearing on the particle surface (E, arrowheads; F). Scale bar, 20 µm. **** p < 0.0001; Welch's t test; n = 27 versus 90 cells. (G) Working model of the PI3K–Rab18‐GDP–dependent SHH‐XLEV secretion pathway. Related to Figure .

    Journal: Journal of Extracellular Biology

    Article Title: Unconventional Secretion of Angiogenic Sonic Hedgehog–Containing Extra‐Large Extracellular Vesicles is Driven by PI3K–Rab18‐GDP Signalling

    doi: 10.1002/jex2.70112

    Figure Lengend Snippet: GDP‐bound Rab18 promotes perinuclear SHH‐XLEV secretion. (A–C) Spinning disk microscopy of NIH3T3 cells coexpressing SHHN‐tagRFP and EGFP‐Rab18 mutants, shown as z‐projections (A), single planes (A), and side views (B), with quantification of perinuclear SHH accumulation (C). Dotted lines, periphery of the Rab18‐GTP‐expressing colony (green) and basolateral SHH deposition (magenta). Arrowheads, colocalization spots of Rab18‐GTP and SHH on the plasma membrane (A) and Rab18‐GTP‐specific basolateral SHH deposition (B). Scale bars, 20 µm. *** p < 0.001; chi‐square test; n = 88–125 cells. Related to . (D) Spinning disk microscopy of NIH3T3 cells undergoing burst‐like secretion of SHHN‐EGFP and Hsp90α‐tagRFP following prolonged CID treatment (arrowheads, 30 h). Scale bar, 20 µm. Related to Figure and . (E, F) Three‐dimensional reconstruction of surface SHH immunofluorescence (red) in SHHN‐EGFP–expressing NIH3T3 cells (E), with quantification of SHH signal intensity appearing on the particle surface (E, arrowheads; F). Scale bar, 20 µm. **** p < 0.0001; Welch's t test; n = 27 versus 90 cells. (G) Working model of the PI3K–Rab18‐GDP–dependent SHH‐XLEV secretion pathway. Related to Figure .

    Article Snippet: EGFP‐Rab18 (Addgene #49550; RRID: Addgene_49550), EGFP‐Rab18Q67L (Addgene #49596; RRID: Addgene_49596), and EGFP‐Rab18S22N (Addgene #49597; RRID: Addgene_49597) were gifts from Marci Scidmore (Huang et al. ).

    Techniques: Microscopy, Expressing, Clinical Proteomics, Membrane, Immunofluorescence

    ( A,B ) Cytoplasmic GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained for GFP under ( A ) permeabilized or ( B ) non-permeabilized conditions. GFP is detected by anti-GFP in permeabilized cells, but not in non-permeabilized cells (asterisks). ( C,D ) SHH-GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained using anti-SHH under, ( C ) permeabilized or, ( D ) non-permeabilized conditions. SHH-GFP signal is evident in cytonemes in both conditions (yellow arrows), but not evident in non-permeabilized cells when probed with anti-SHH (red arrows). ( E–G ) Cytonemes of NIH3T3 cells expressing SHH-mCherry and CD63-EGFP ( E ) or Rab18-EGFP ( G ) analyzed by confocal microscopy do not show significant co-localization. Rab18-EGFP does not enter cytonemes. ( F ) Box plot with min/max whiskers of Pearson’s coefficients values of colocalization between SHH-mCherry and CD63-EGFP in cytonemes.

    Journal: eLife

    Article Title: Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex

    doi: 10.7554/eLife.61432

    Figure Lengend Snippet: ( A,B ) Cytoplasmic GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained for GFP under ( A ) permeabilized or ( B ) non-permeabilized conditions. GFP is detected by anti-GFP in permeabilized cells, but not in non-permeabilized cells (asterisks). ( C,D ) SHH-GFP and mCherry-Mem transfected NIH3T3 cells were immuno-stained using anti-SHH under, ( C ) permeabilized or, ( D ) non-permeabilized conditions. SHH-GFP signal is evident in cytonemes in both conditions (yellow arrows), but not evident in non-permeabilized cells when probed with anti-SHH (red arrows). ( E–G ) Cytonemes of NIH3T3 cells expressing SHH-mCherry and CD63-EGFP ( E ) or Rab18-EGFP ( G ) analyzed by confocal microscopy do not show significant co-localization. Rab18-EGFP does not enter cytonemes. ( F ) Box plot with min/max whiskers of Pearson’s coefficients values of colocalization between SHH-mCherry and CD63-EGFP in cytonemes.

    Article Snippet: transfected construct ( Homo sapiens ) , pCMV-EGFP-Rab18 , Addgene , RRID: Addgene_49550 , Marci Scidmore.

    Techniques: Transfection, Staining, Expressing, Confocal Microscopy

    Journal: eLife

    Article Title: Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex

    doi: 10.7554/eLife.61432

    Figure Lengend Snippet:

    Article Snippet: transfected construct ( Homo sapiens ) , pCMV-EGFP-Rab18 , Addgene , RRID: Addgene_49550 , Marci Scidmore.

    Techniques: Derivative Assay, Transfection, Construct, Recombinant, In Situ, Software, Imaging, Luciferase, Reporter Assay, Western Blot