dope  (Echelon Biosciences)


Bioz Verified Symbol Echelon Biosciences is a verified supplier
Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences dope
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    93/100 stars

    Images

    dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Echelon Biosciences dope
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    86/100 stars

    Images

    atto647n dope atto tec  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Echelon Biosciences atto647n dope atto tec
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Atto647n Dope Atto Tec, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atto647n dope atto tec/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atto647n dope atto tec - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion"

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    Journal: Current Biology

    doi: 10.1016/j.cub.2022.04.089

    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Figure Legend Snippet: Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Techniques Used: Binding Assay, Generated, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .
    Figure Legend Snippet: Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Techniques Used: Transfection, Staining, Purification, Recombinant, Labeling, Standard Deviation, Expressing, Confocal Microscopy

    rhodamine dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Echelon Biosciences rhodamine dope
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped <t>with</t> <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Rhodamine Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhodamine dope/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhodamine dope - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion"

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    Journal: Current Biology

    doi: 10.1016/j.cub.2022.04.089

    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Figure Legend Snippet: Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Techniques Used: Binding Assay, Generated, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .
    Figure Legend Snippet: Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Techniques Used: Transfection, Staining, Purification, Recombinant, Labeling, Standard Deviation, Expressing, Confocal Microscopy

    atto647n dope atto tec  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Echelon Biosciences atto647n dope atto tec
    A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.
    Atto647n Dope Atto Tec, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atto647n dope atto tec/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atto647n dope atto tec - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion"

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

    Journal: bioRxiv

    doi: 10.1101/2021.10.14.464363

    A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.
    Figure Legend Snippet: A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.

    Techniques Used: Generated, Binding Assay, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    A-B , NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background), n=3-4 repeats with 2936 individual cells each. Asterisks denote p<0.001. Scale bar, 10 μm, inset, 1 μm. C , Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. D , Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labelled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n=92-99 beads. Scale bars, 10 μm. E , NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n=3 repeats with 7-25 individual cells each, asterisks denote p<0.001.
    Figure Legend Snippet: A-B , NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background), n=3-4 repeats with 2936 individual cells each. Asterisks denote p<0.001. Scale bar, 10 μm, inset, 1 μm. C , Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. D , Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labelled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n=92-99 beads. Scale bars, 10 μm. E , NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n=3 repeats with 7-25 individual cells each, asterisks denote p<0.001.

    Techniques Used: Transfection, Staining, Purification, Recombinant, Standard Deviation, Expressing, Confocal Microscopy

    rhodamine dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Echelon Biosciences rhodamine dope
    A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped <t>with</t> <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.
    Rhodamine Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhodamine dope/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhodamine dope - by Bioz Stars, 2023-01
    86/100 stars

    Images

    1) Product Images from "A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion"

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C driven phosphoinositide conversion

    Journal: bioRxiv

    doi: 10.1101/2021.10.14.464363

    A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.
    Figure Legend Snippet: A , Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently-tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. B , Reconstituted fluorescently-tagged Subcomplex-1, −2, or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. C , Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877-1395 segmented beads per condition. D-F , Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplex-1, −2, or holocomplex were added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. 340-1359 beads were quantified per concentration, mean±standard deviation. G , Schematic of liposome tethering experiment. Subcomplex-1, −2, or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. H-I , Membranes and exocyst complexes were imaged by confocal microscopy, segmented and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430-778 individual beads. MFI, mean fluorescent intensity. J , Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other.

    Techniques Used: Generated, Binding Assay, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    A-B , NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background), n=3-4 repeats with 2936 individual cells each. Asterisks denote p<0.001. Scale bar, 10 μm, inset, 1 μm. C , Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. D , Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labelled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n=92-99 beads. Scale bars, 10 μm. E , NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n=3 repeats with 7-25 individual cells each, asterisks denote p<0.001.
    Figure Legend Snippet: A-B , NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background), n=3-4 repeats with 2936 individual cells each. Asterisks denote p<0.001. Scale bar, 10 μm, inset, 1 μm. C , Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. D , Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labelled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n=92-99 beads. Scale bars, 10 μm. E , NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n=3 repeats with 7-25 individual cells each, asterisks denote p<0.001.

    Techniques Used: Transfection, Staining, Purification, Recombinant, Standard Deviation, Expressing, Confocal Microscopy

    dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences dope
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    93/100 stars

    Images

    dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences dope
    PI(3)P-dependent lipid binding of hSNX16 CC domain mutants. (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PX-CC dimer (PDB accession number 5GW0 ; ). CC domains are shown in green and glutamates in magenta. (C–F) Liposome cosedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in C and E. (C and D) Liposomes composed of <t>80%</t> <t>DOPC</t> (1,2-dioleoyl-sn-glycero-3-phosphocholine), 15% <t>DOPE</t> (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 5% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in DOPC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild-type hSNX16. Y145A mutsation abolishes PI(3)P binding of hSNX16 as previously reported . (E and F) Binding of wild-type hSNX16 is more salt sensitive than hSNX16 3A . Liposomes (70% DOPC, 15% DOPE, 5% DOPS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data are presented as mean ± SEM. *, P < 0.05. ns, not significant.
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    93/100 stars

    Images

    1) Product Images from "Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes"

    Article Title: Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201811074

    PI(3)P-dependent lipid binding of hSNX16 CC domain mutants. (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PX-CC dimer (PDB accession number 5GW0 ; ). CC domains are shown in green and glutamates in magenta. (C–F) Liposome cosedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in C and E. (C and D) Liposomes composed of 80% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 15% DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 5% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in DOPC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild-type hSNX16. Y145A mutsation abolishes PI(3)P binding of hSNX16 as previously reported . (E and F) Binding of wild-type hSNX16 is more salt sensitive than hSNX16 3A . Liposomes (70% DOPC, 15% DOPE, 5% DOPS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data are presented as mean ± SEM. *, P < 0.05. ns, not significant.
    Figure Legend Snippet: PI(3)P-dependent lipid binding of hSNX16 CC domain mutants. (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PX-CC dimer (PDB accession number 5GW0 ; ). CC domains are shown in green and glutamates in magenta. (C–F) Liposome cosedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in C and E. (C and D) Liposomes composed of 80% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 15% DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), 5% DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in DOPC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild-type hSNX16. Y145A mutsation abolishes PI(3)P binding of hSNX16 as previously reported . (E and F) Binding of wild-type hSNX16 is more salt sensitive than hSNX16 3A . Liposomes (70% DOPC, 15% DOPE, 5% DOPS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. Data are presented as mean ± SEM. *, P < 0.05. ns, not significant.

    Techniques Used: Binding Assay, Construct, Purification, Incubation, Staining, Concentration Assay

    dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences dope
    (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PXCC dimer (PDB# 5gw0; . CC domains are shown in green, and glutamates in magenta. (C-F) Liposome co-sedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in (C) and (E). (C, D) Liposomes composed of <t>80%</t> <t>DOPC,</t> 15% <t>DOPE,</t> 5% DOPS, and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in PC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild type hSNX16. Y145A mutation abolishes PI(3)P binding of hSNX16 as previously reported . (E, F) Binding of wild type hSNX16 is more salt-sensitive than hSNX16 3A . Liposomes (70% PC, 15% PE, 5% PS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data are represented as mean ± SEM. *p < 0.05.
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    93/100 stars

    Images

    1) Product Images from "Higher order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes"

    Article Title: Higher order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes

    Journal: bioRxiv

    doi: 10.1101/469932

    (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PXCC dimer (PDB# 5gw0; . CC domains are shown in green, and glutamates in magenta. (C-F) Liposome co-sedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in (C) and (E). (C, D) Liposomes composed of 80% DOPC, 15% DOPE, 5% DOPS, and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in PC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild type hSNX16. Y145A mutation abolishes PI(3)P binding of hSNX16 as previously reported . (E, F) Binding of wild type hSNX16 is more salt-sensitive than hSNX16 3A . Liposomes (70% PC, 15% PE, 5% PS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data are represented as mean ± SEM. *p < 0.05.
    Figure Legend Snippet: (A) Schematic of hSNX16 constructs. (B) Location of hSNX16 CC mutations on hSNX16 PXCC dimer (PDB# 5gw0; . CC domains are shown in green, and glutamates in magenta. (C-F) Liposome co-sedimentation assays. Purified hSNX16 variants were incubated with liposomes of the indicated composition and pelleted. Representative Coomassie staining of supernatant (S) and pellet (P) fractions is shown in (C) and (E). (C, D) Liposomes composed of 80% DOPC, 15% DOPE, 5% DOPS, and PI(3)P (0.5%, 1%, 2.5%, 5%, and 10%; with a corresponding decrease in PC). PI(3)P-dependent lipid binding of purified hSNX16 CC domain mutants is not significantly different from wild type hSNX16. Y145A mutation abolishes PI(3)P binding of hSNX16 as previously reported . (E, F) Binding of wild type hSNX16 is more salt-sensitive than hSNX16 3A . Liposomes (70% PC, 15% PE, 5% PS, and 10% PI(3)P) were incubated for 45 min with purified hSNX16 and the indicated NaCl concentrations before pelleting. In the last condition, hSNX16 and liposomes were incubated in 100 mM NaCl for 30 min, and then NaCl was added to a final concentration of 400 mM for 15 min before pelleting. Quantification is a result of three independent experiments, analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Data are represented as mean ± SEM. *p < 0.05.

    Techniques Used: Construct, Sedimentation, Purification, Incubation, Staining, Binding Assay, Mutagenesis, Concentration Assay

    dioleoyl sn glycero 3 phosphoethanolamine dope  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences dioleoyl sn glycero 3 phosphoethanolamine dope
    Dioleoyl Sn Glycero 3 Phosphoethanolamine Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dioleoyl sn glycero 3 phosphoethanolamine dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dioleoyl sn glycero 3 phosphoethanolamine dope - by Bioz Stars, 2023-01
    93/100 stars

    Images

    l 2182 pi  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
    Bioz Manufacturer Symbol Echelon Biosciences manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Echelon Biosciences l 2182 pi
    L 2182 Pi, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l 2182 pi/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l 2182 pi - by Bioz Stars, 2023-01
    93/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Echelon Biosciences dope
    Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dope - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    86
    Echelon Biosciences atto647n dope atto tec
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Atto647n Dope Atto Tec, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atto647n dope atto tec/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atto647n dope atto tec - by Bioz Stars, 2023-01
    86/100 stars
      Buy from Supplier

    86
    Echelon Biosciences rhodamine dope
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped <t>with</t> <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Rhodamine Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rhodamine dope/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rhodamine dope - by Bioz Stars, 2023-01
    86/100 stars
      Buy from Supplier

    93
    Echelon Biosciences dioleoyl sn glycero 3 phosphoethanolamine dope
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped <t>with</t> <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    Dioleoyl Sn Glycero 3 Phosphoethanolamine Dope, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dioleoyl sn glycero 3 phosphoethanolamine dope/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dioleoyl sn glycero 3 phosphoethanolamine dope - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    93
    Echelon Biosciences l 2182 pi
    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped <t>with</t> <t>Atto647N-DOPE</t> and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .
    L 2182 Pi, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l 2182 pi/product/Echelon Biosciences
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l 2182 pi - by Bioz Stars, 2023-01
    93/100 stars
      Buy from Supplier

    Image Search Results


    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Journal: Current Biology

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    doi: 10.1016/j.cub.2022.04.089

    Figure Lengend Snippet: Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Article Snippet: Liposomes containing either PI(4)P (Echelon Biosciences) or PI(4,5)P 2 (Echelon Biosciences) were produced by mixing 85 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10 mol % phosphatidylserine (POPS) with 5 mol % of the respective phosphatidylinositol together with 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE.

    Techniques: Binding Assay, Generated, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Journal: Current Biology

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    doi: 10.1016/j.cub.2022.04.089

    Figure Lengend Snippet: Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Article Snippet: Liposomes containing either PI(4)P (Echelon Biosciences) or PI(4,5)P 2 (Echelon Biosciences) were produced by mixing 85 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10 mol % phosphatidylserine (POPS) with 5 mol % of the respective phosphatidylinositol together with 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE.

    Techniques: Transfection, Staining, Purification, Recombinant, Labeling, Standard Deviation, Expressing, Confocal Microscopy

    Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Journal: Current Biology

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    doi: 10.1016/j.cub.2022.04.089

    Figure Lengend Snippet: Independent binding of exocyst subcomplexes-1 and -2 to PI(4,5)P 2 enables holocomplex tethering (A) Schematic of phosphoinositide specificity experiment. Membrane-coated beads of distinct lipid composition were generated. To these beads, fluorescently tagged exocyst subcomplexes were added to assess membrane binding by fluorescence microscopy. (B) Reconstituted fluorescently tagged subcomplexes-1 and -2 or holocomplex were added at 100 nM to membrane-coated beads. These were formed from a lipid composition of 84.9% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10% phosphatidylserine, and 5% phosphoinositide or POPC, doped with 0.1% rhodamine-DPPE. Beads were imaged by confocal microscopy. Scale bar, 10 μm. (C) Membrane-coated beads were segmented using the rhodamine lipid signal as a membrane mask, and the mean fluorescence intensities of exocyst complexes bound to each bead were determined. Violin plots, 877–1,395 segmented beads per condition. (D–F) Relative PI(4,5)P 2 membrane binding affinities for the exocyst complexes. Subcomplexes-1 and -2 or holocomplex was added in a dilution series to membrane-coated beads containing 5% PI(4,5)P 2 and imaged by confocal microscopy. Individual beads were segmented, and a binding saturation curve was fitted resulting in mean kD determination with 95% confidence intervals given. In total, 340–1,359 beads were quantified per concentration, mean ± standard deviation. (G) Schematic of liposome tethering experiment. Subcomplexes-1 and -2 or holocomplex (150 nM) were bound to 5% PI(4,5)P 2 membranes. Liposomes doped with Atto647N-DOPE and composed with either 5% PI(4)P or PI(4,5)P 2 were added. (H and I) Membranes and exocyst complexes were imaged by confocal microscopy, segmented, and the fluorescent intensity of tethered liposomes was measured. Violin plots from 430–778 individual beads. MFI, mean fluorescent intensity. Scale bar, 10 μm. (J) Exocyst holocomplex tethers two PI(4,5)P 2 , but not PI(4)P, containing membranes to each other. See also .

    Article Snippet: Liposomes containing either PI(4)P (Echelon Biosciences) or PI(4,5)P 2 (Echelon Biosciences) were produced by mixing 85 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10 mol % phosphatidylserine (POPS) with 5 mol % of the respective phosphatidylinositol together with 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE.

    Techniques: Binding Assay, Generated, Fluorescence, Microscopy, Confocal Microscopy, Concentration Assay, Standard Deviation

    Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Journal: Current Biology

    Article Title: A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion

    doi: 10.1016/j.cub.2022.04.089

    Figure Lengend Snippet: Arf6 controls PI(4,5)P 2 conversion and exocyst recruitment (A and B) NMuMG cells were transfected with Arf6-Q67L or Arf6-T27N tagged with mRuby3, and subsequently fixed, permeabilized, and stained against either PI(4)P or PI(4,5)P 2 using purified SidC or PLCδ-PH fused to GFP, respectively. Signal overlap between channels was determined using the ImageJ plugin Squassh. Plot shows biological repeats (in bold) with individual cells (in background); n = 3–4 repeats with 29–36 individual cells each. Asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. (C) Purified GST, GST-Arf6-T27N or GST-Arf6-Q67L were bound to resin as bait, and purified PIP5K1C (400 nM) used as prey. Inset (top) is increased contrast. (D) Membrane-coated beads harboring PI(4)P and either recombinant Arf6-T27N or Arf6-Q67L were distinctly labeled with NBD-DPPE or Atto647N-DOPE and mixed. PIP5K1C (12.5 nM) was added, and the recruitment of purified PLCδ-PH fused to RFP was measured over time. Beads were segmented using either the NBD or Atto647N signal as masks. Plot is mean fluorescent intensity of recruited PLCδ-PH ± standard deviation, quantified in ImageJ. n = 92–99 beads. Scale bar, 10 μm. (E) NMuMG cells expressing endogenously sfGFP-tagged Exoc4 were transfected with Arf6-Q67L or Arf6-T27N fused to mRuby3 and imaged using Airyscan confocal microscopy. Signal overlap between channels was determined using the ImageJ plugin Squassh. n = 3 repeats with 7–25 individual cells each; asterisks denote p < 0.001. Scale bar, 10 μm; inset, 1 μm. See also .

    Article Snippet: Liposomes containing either PI(4)P (Echelon Biosciences) or PI(4,5)P 2 (Echelon Biosciences) were produced by mixing 85 mol % 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 10 mol % phosphatidylserine (POPS) with 5 mol % of the respective phosphatidylinositol together with 0.1% Atto647N-DOPE (ATTO-TEC) or Rhodamine-DOPE.

    Techniques: Transfection, Staining, Purification, Recombinant, Labeling, Standard Deviation, Expressing, Confocal Microscopy