bodipy tmr pip3  (Echelon Biosciences)


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    Echelon Biosciences bodipy tmr pip3
    Bodipy Tmr Pip3, 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
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    bodipy fl phosphatidylinositol  (Echelon Biosciences)


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    Echelon Biosciences bodipy fl phosphatidylinositol
    Bodipy Fl Phosphatidylinositol, 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
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    fluorescent bodipy pi 3 p  (Echelon Biosciences)


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    Echelon Biosciences fluorescent bodipy pi 3 p
    (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with <t>PI(3)P,</t> and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).
    Fluorescent Bodipy Pi 3 P, 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
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    Images

    1) Product Images from "ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes"

    Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

    Journal: bioRxiv

    doi: 10.1101/2023.03.22.533832

    (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with PI(3)P, and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).
    Figure Legend Snippet: (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with PI(3)P, and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).

    Techniques Used: Binding Assay, Fluorescence, Recombinant, Incubation

    (A) Still images from a lipid FRAP series of BODIPY-PI(3)P in GUVs in the presence of 100 µM Ca 2+ with and without 0.5 µM ANXA11 FL. Scale Bar - 5 µm. (B) PI(3)P fluorescence recovery rates of GUVs in the presence of 100 µM Ca 2+ co-incubated with either 0.5 µM ANXA11 FL or 0.5 µM ARD. The % fluorescence recovery by 5 sec is plotted alongside. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ns - not significant (p > 0.05), n=4. (C) A schematic illustrating the change in PK dye fluorescence emission as lipids transition from a disordered (red emission) to ordered (blue emission) state. (D) Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative lipid order (φ) of GUVs alone or with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or 0.5 µM ARD. Scale Bar - 5 µm. (E) Quantification of the relative lipid order (φ) of GUVs as shown in (D), including protein alone and Ca 2+ alone controls. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant, n=3 (30-82 GUVs). (F) A schematic of our AFM-IR setup which acquires nanoscale resolved chemical spectra of protein and lipid components. (G) An exemplary AFM-IR morphology map of GUV fragments bound to ANXA11. Green crosses indicate regions with a ‘lipid only’ signature. Red crosses indicate regions with ‘protein and lipid’ signatures. Scale bars - 2 µm. (H) A ratio of the infrared maps from protein:lipid (1655/1730 cm -1 ) spectroscopic signatures illustrates the heterogeneity of their spatial distribution at nanoscale. Scale bars - 2 µm. (I) A comparison of the average (Mean ± SD) AFM-IR spectra of GUV fragments bound to 0.5 µM ANXA11 FL and 0.5 µM ARD at 100 µM Ca 2+ . 450 spectra were collected from across 69 independent GUV fragments (12 lipid only, 31 lipid+FL, 26 lipid+ARD) across 5 experimental repeats. The SD in the ARD condition falls beneath the thickness of the line. (J) Second derivatives of the spectra (Mean ± SD) in the IR absorption of the C=O stretching region of lipids for GUV fragments alone or bound to either ANXA11 FL or ARD. (K) Quantification of the wavenumber of GUV fragments alone compared with GUV fragments bound to ANXA11 FL or ARD. The ∼1730 to ∼1738 cm -1 shift indicates a liquid-to-gel lipid phase transition. Mean ± SD and 25-75 th percentile (box). One-way ANOVA with Tukey’s multiple comparison, ***p < 0.001, ns - not significant (p > 0.05), n=5 (12-26 GUV fragments).
    Figure Legend Snippet: (A) Still images from a lipid FRAP series of BODIPY-PI(3)P in GUVs in the presence of 100 µM Ca 2+ with and without 0.5 µM ANXA11 FL. Scale Bar - 5 µm. (B) PI(3)P fluorescence recovery rates of GUVs in the presence of 100 µM Ca 2+ co-incubated with either 0.5 µM ANXA11 FL or 0.5 µM ARD. The % fluorescence recovery by 5 sec is plotted alongside. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ns - not significant (p > 0.05), n=4. (C) A schematic illustrating the change in PK dye fluorescence emission as lipids transition from a disordered (red emission) to ordered (blue emission) state. (D) Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative lipid order (φ) of GUVs alone or with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or 0.5 µM ARD. Scale Bar - 5 µm. (E) Quantification of the relative lipid order (φ) of GUVs as shown in (D), including protein alone and Ca 2+ alone controls. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant, n=3 (30-82 GUVs). (F) A schematic of our AFM-IR setup which acquires nanoscale resolved chemical spectra of protein and lipid components. (G) An exemplary AFM-IR morphology map of GUV fragments bound to ANXA11. Green crosses indicate regions with a ‘lipid only’ signature. Red crosses indicate regions with ‘protein and lipid’ signatures. Scale bars - 2 µm. (H) A ratio of the infrared maps from protein:lipid (1655/1730 cm -1 ) spectroscopic signatures illustrates the heterogeneity of their spatial distribution at nanoscale. Scale bars - 2 µm. (I) A comparison of the average (Mean ± SD) AFM-IR spectra of GUV fragments bound to 0.5 µM ANXA11 FL and 0.5 µM ARD at 100 µM Ca 2+ . 450 spectra were collected from across 69 independent GUV fragments (12 lipid only, 31 lipid+FL, 26 lipid+ARD) across 5 experimental repeats. The SD in the ARD condition falls beneath the thickness of the line. (J) Second derivatives of the spectra (Mean ± SD) in the IR absorption of the C=O stretching region of lipids for GUV fragments alone or bound to either ANXA11 FL or ARD. (K) Quantification of the wavenumber of GUV fragments alone compared with GUV fragments bound to ANXA11 FL or ARD. The ∼1730 to ∼1738 cm -1 shift indicates a liquid-to-gel lipid phase transition. Mean ± SD and 25-75 th percentile (box). One-way ANOVA with Tukey’s multiple comparison, ***p < 0.001, ns - not significant (p > 0.05), n=5 (12-26 GUV fragments).

    Techniques Used: Fluorescence, Incubation, Sublimation

    bodipy tmr phosphatidylinositol 4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences bodipy tmr phosphatidylinositol 4 5 bisphosphate
    KEY RESOURCES TABLE
    Bodipy Tmr Phosphatidylinositol 4 5 Bisphosphate, 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
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    1) Product Images from "Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics"

    Article Title: Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics

    Journal: Cell

    doi: 10.1016/j.cell.2022.10.018


    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Electrophoresis, Expressing, Plasmid Preparation, cAMP Assay, Clone Assay, Software, Flow Cytometry

    bodipy tmr  (Echelon Biosciences)


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    Echelon Biosciences bodipy tmr
    KEY RESOURCES TABLE
    Bodipy Tmr, 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
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    1) Product Images from "Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics"

    Article Title: Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics

    Journal: Cell

    doi: 10.1016/j.cell.2022.10.018

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Electrophoresis, Expressing, Plasmid Preparation, cAMP Assay, Clone Assay, Software, Flow Cytometry

    bodipy fl phosphatidylinositol  (Echelon Biosciences)


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    Echelon Biosciences bodipy fl phosphatidylinositol
    Bodipy Fl Phosphatidylinositol, 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
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    bodipy labeled phosphatidylinositol 4 5 phosphate fl pip2  (Echelon Biosciences)


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    Echelon Biosciences bodipy labeled phosphatidylinositol 4 5 phosphate fl pip2
    Bodipy Labeled Phosphatidylinositol 4 5 Phosphate Fl Pip2, 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
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    c 05r16 bodipy pi5p  (Echelon Biosciences)


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    Echelon Biosciences c 05r16 bodipy pi5p
    Table 1
    C 05r16 Bodipy Pi5p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes"

    Article Title: Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes

    Journal: Plant Signaling & Behavior

    doi:

    Table 1
    Figure Legend Snippet: Table 1

    Techniques Used:

    Table 2
    Figure Legend Snippet: Table 2

    Techniques Used: Expressing

    Table 3
    Figure Legend Snippet: Table 3

    Techniques Used:

    c 05r16 bodipy pi5p  (Echelon Biosciences)


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    Echelon Biosciences c 05r16 bodipy pi5p
    Table 1
    C 05r16 Bodipy Pi5p, 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/c 05r16 bodipy pi5p/product/Echelon Biosciences
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    1) Product Images from "Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes"

    Article Title: Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes

    Journal: Plant Signaling & Behavior

    doi:

    Table 1
    Figure Legend Snippet: Table 1

    Techniques Used:

    Table 2
    Figure Legend Snippet: Table 2

    Techniques Used: Expressing

    Table 3
    Figure Legend Snippet: Table 3

    Techniques Used:

    bodipy fl pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences bodipy fl pi 4 5 p 2
    A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.
    Bodipy Fl Pi 4 5 P 2, 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
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    1) Product Images from "Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P 2 -Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing"

    Article Title: Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P 2 -Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103127

    A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.
    Figure Legend Snippet: A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.

    Techniques Used: Incubation, Infection, Staining, Colony-forming Unit Assay, Lactate Dehydrogenase Assay, Lysis

    A ) Enzymatic activity of rExoU on BODIPY-FL-PI(4,5)P 2 determined by TLC. The fluorescent substrate was incubated with 13.5 or 6.76 pmol rExoU in the presence of the cofactor ubiquitin (13.5 pmol), labeled as U + Ub. PI-PLC and honeybee PLA 2 , 13.5 pmol each, were also tested. The position of the non-hydrolyzed substrate is indicated with an arrow. The percentage hydrolysis of each enzyme, averaged from 3 independent experiments, is shown below the chromatograph. B ) Localization of ExoU-S142A in HeLa cells after infection in the presence or absence of the extracellular phospholipids. Infected cells were harvested, mechanically lysed, and fractionated. Fractions were analyzed by Western blot using an anti-ExoU monoclonal antibody. S: soluble fraction, M: membrane fraction. To achieve a linear detection range, 4-fold higher volumes of membrane fractions were loaded relative to soluble material. C ) The Western blot signal intensity of both unmodified and ubiquitinated ExoU-S142A was quantified. Membrane fraction (dark gray) and soluble fraction (light gray) are shown as a 100% stacked column chart with the mean ± SD from 3 independent experiments. D ) rExoU (13.5 pmol) binding to phospholipid (1600 pmol)-coated polystyrene plates detected by fluorescence microscopy. Magnification: 40x. E ) rExoU affinity to phospholipids evaluated by an ELISA-based solid-phase binding assay. Polystyrene plates were coated with the indicated amounts of phospholipids. The binding of 13.5 pmol rExoU was determined using an anti-ExoU antibody and a horseradish peroxidase-conjugated secondary antibody. Peroxidase activity on the substrate ABTS with H 2 O 2 was measured by absorbance at 405 nm. F ) The Kd of rExoU to PI(4,5)P 2 determined with the ELISA-based binding assay. Nonlinear regression analysis of the concentration of rExoU (nM) as a function of total binding is shown. The plot represents the mean ± SEM of duplicates from 2 independent experiments. G ) Binding of the PLCδ1-PH domain to PI(4,5)P 2 evaluated by nonlinear regression of the concentration of the PLCδ1-PH peptide (nM) as a function of total binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments. H ) Inhibitory effect of rExoU to PLCδ1-PH binding to PI(4,5)P 2 . rExoU (between 6.25 and 800 nM) was mixed into 50 nM of the PLCδ1-PH peptide for the solid-phase binding assay. IC 50 : the concentration of rExoU to displace 50% of PLCδ1-PH binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments.
    Figure Legend Snippet: A ) Enzymatic activity of rExoU on BODIPY-FL-PI(4,5)P 2 determined by TLC. The fluorescent substrate was incubated with 13.5 or 6.76 pmol rExoU in the presence of the cofactor ubiquitin (13.5 pmol), labeled as U + Ub. PI-PLC and honeybee PLA 2 , 13.5 pmol each, were also tested. The position of the non-hydrolyzed substrate is indicated with an arrow. The percentage hydrolysis of each enzyme, averaged from 3 independent experiments, is shown below the chromatograph. B ) Localization of ExoU-S142A in HeLa cells after infection in the presence or absence of the extracellular phospholipids. Infected cells were harvested, mechanically lysed, and fractionated. Fractions were analyzed by Western blot using an anti-ExoU monoclonal antibody. S: soluble fraction, M: membrane fraction. To achieve a linear detection range, 4-fold higher volumes of membrane fractions were loaded relative to soluble material. C ) The Western blot signal intensity of both unmodified and ubiquitinated ExoU-S142A was quantified. Membrane fraction (dark gray) and soluble fraction (light gray) are shown as a 100% stacked column chart with the mean ± SD from 3 independent experiments. D ) rExoU (13.5 pmol) binding to phospholipid (1600 pmol)-coated polystyrene plates detected by fluorescence microscopy. Magnification: 40x. E ) rExoU affinity to phospholipids evaluated by an ELISA-based solid-phase binding assay. Polystyrene plates were coated with the indicated amounts of phospholipids. The binding of 13.5 pmol rExoU was determined using an anti-ExoU antibody and a horseradish peroxidase-conjugated secondary antibody. Peroxidase activity on the substrate ABTS with H 2 O 2 was measured by absorbance at 405 nm. F ) The Kd of rExoU to PI(4,5)P 2 determined with the ELISA-based binding assay. Nonlinear regression analysis of the concentration of rExoU (nM) as a function of total binding is shown. The plot represents the mean ± SEM of duplicates from 2 independent experiments. G ) Binding of the PLCδ1-PH domain to PI(4,5)P 2 evaluated by nonlinear regression of the concentration of the PLCδ1-PH peptide (nM) as a function of total binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments. H ) Inhibitory effect of rExoU to PLCδ1-PH binding to PI(4,5)P 2 . rExoU (between 6.25 and 800 nM) was mixed into 50 nM of the PLCδ1-PH peptide for the solid-phase binding assay. IC 50 : the concentration of rExoU to displace 50% of PLCδ1-PH binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments.

    Techniques Used: Activity Assay, Incubation, Labeling, Infection, Western Blot, Binding Assay, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Concentration Assay

    A ) After translocation into the host cytoplasm by T3SS, ExoU (U) is postulated to bind to PI(4,5)P 2 residing on the inner leaflet of the plasma membrane. The PLA 2 activity of ExoU is activated by a cofactor (Co), ubiquitin or ubiquitinated proteins. B ) ExoU hydrolyzes PI(4,5)P 2 and/or other phospholipid species (shown as Xs), leaving lyso-phospholipids, destabilizing the membrane. Reduction of PI(4,5)P 2 disrupts the anchoring and interaction between the focal adhesion, cytoskeletal structure, and plasma membrane, possibly through a cell signaling pathway. Cells round due to the weakened adhesion capability and actin filament depolymerization, leading to cytoskeletal collapse. The outer leaflet of the plasma membrane is intact at this stage. C ) At a middle stage of infection, the plasma membrane starts to bleb, facilitating further membrane damage. D ) During a late stage of infection, the outer leaflet of the plasma membrane is compromised, allowing the influx of an impermeable nucleic acid dye and staining of the nucleus (green), consequently large molecules (e.g. LDH) are released from lysed cells.
    Figure Legend Snippet: A ) After translocation into the host cytoplasm by T3SS, ExoU (U) is postulated to bind to PI(4,5)P 2 residing on the inner leaflet of the plasma membrane. The PLA 2 activity of ExoU is activated by a cofactor (Co), ubiquitin or ubiquitinated proteins. B ) ExoU hydrolyzes PI(4,5)P 2 and/or other phospholipid species (shown as Xs), leaving lyso-phospholipids, destabilizing the membrane. Reduction of PI(4,5)P 2 disrupts the anchoring and interaction between the focal adhesion, cytoskeletal structure, and plasma membrane, possibly through a cell signaling pathway. Cells round due to the weakened adhesion capability and actin filament depolymerization, leading to cytoskeletal collapse. The outer leaflet of the plasma membrane is intact at this stage. C ) At a middle stage of infection, the plasma membrane starts to bleb, facilitating further membrane damage. D ) During a late stage of infection, the outer leaflet of the plasma membrane is compromised, allowing the influx of an impermeable nucleic acid dye and staining of the nucleus (green), consequently large molecules (e.g. LDH) are released from lysed cells.

    Techniques Used: Translocation Assay, Activity Assay, Infection, Staining

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    Echelon Biosciences bodipy tmr pip3
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    (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with <t>PI(3)P,</t> and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).
    Fluorescent Bodipy Pi 3 P, 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
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    Echelon Biosciences bodipy fl pi 4 5 p 2
    A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.
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    Image Search Results


    (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with PI(3)P, and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).

    Journal: bioRxiv

    Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

    doi: 10.1101/2023.03.22.533832

    Figure Lengend Snippet: (A) A schematic of full length (FL) ANXA11 binding to lysosomal membranes, illustrating the Ca 2+ -dependent annexin repeat domain (ARD) association with PI(3)P, and the cytosolic-facing low complexity domain (LCD). (B) Fluorescence micrographs of recombinant AF647-labelled FL (aa 1-502), LCD (aa 1-185) and ARD (aa 186-502) ANXA11 at varying protein concentrations. Scale bar – 5 µm. (C) Representative fluorescence images of ATTO488 GUVs incubated with 0.5 µM AF555-labelled ANXA11 FL, LCD and ARD in the presence or absence of 500 µM Ca 2+ . Scale bar – 5 µm. (D) Quantification of the fluorescence intensity of AF555-labelled FL, LCD and ARD recruited to GUVs as shown in (C) at varying Ca 2+ concentrations. Mean ± SD. Kruskal-Wallis test with Dunn’s multiple comparison, ***p < 0.001, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (110-379 GUVs).

    Article Snippet: To test the effect of ANXA11 on lipid mobility, 20 μM GUVs in which the PI(3)P was replaced with fluorescent BODIPY-PI(3)P (Echelon) were incubated with or without 0.5 μM AF647-labelled ANXA11 FL for 15-30 min at 37 °C in 100 μM Ca 2+ as described above.

    Techniques: Binding Assay, Fluorescence, Recombinant, Incubation

    (A) Still images from a lipid FRAP series of BODIPY-PI(3)P in GUVs in the presence of 100 µM Ca 2+ with and without 0.5 µM ANXA11 FL. Scale Bar - 5 µm. (B) PI(3)P fluorescence recovery rates of GUVs in the presence of 100 µM Ca 2+ co-incubated with either 0.5 µM ANXA11 FL or 0.5 µM ARD. The % fluorescence recovery by 5 sec is plotted alongside. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ns - not significant (p > 0.05), n=4. (C) A schematic illustrating the change in PK dye fluorescence emission as lipids transition from a disordered (red emission) to ordered (blue emission) state. (D) Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative lipid order (φ) of GUVs alone or with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or 0.5 µM ARD. Scale Bar - 5 µm. (E) Quantification of the relative lipid order (φ) of GUVs as shown in (D), including protein alone and Ca 2+ alone controls. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant, n=3 (30-82 GUVs). (F) A schematic of our AFM-IR setup which acquires nanoscale resolved chemical spectra of protein and lipid components. (G) An exemplary AFM-IR morphology map of GUV fragments bound to ANXA11. Green crosses indicate regions with a ‘lipid only’ signature. Red crosses indicate regions with ‘protein and lipid’ signatures. Scale bars - 2 µm. (H) A ratio of the infrared maps from protein:lipid (1655/1730 cm -1 ) spectroscopic signatures illustrates the heterogeneity of their spatial distribution at nanoscale. Scale bars - 2 µm. (I) A comparison of the average (Mean ± SD) AFM-IR spectra of GUV fragments bound to 0.5 µM ANXA11 FL and 0.5 µM ARD at 100 µM Ca 2+ . 450 spectra were collected from across 69 independent GUV fragments (12 lipid only, 31 lipid+FL, 26 lipid+ARD) across 5 experimental repeats. The SD in the ARD condition falls beneath the thickness of the line. (J) Second derivatives of the spectra (Mean ± SD) in the IR absorption of the C=O stretching region of lipids for GUV fragments alone or bound to either ANXA11 FL or ARD. (K) Quantification of the wavenumber of GUV fragments alone compared with GUV fragments bound to ANXA11 FL or ARD. The ∼1730 to ∼1738 cm -1 shift indicates a liquid-to-gel lipid phase transition. Mean ± SD and 25-75 th percentile (box). One-way ANOVA with Tukey’s multiple comparison, ***p < 0.001, ns - not significant (p > 0.05), n=5 (12-26 GUV fragments).

    Journal: bioRxiv

    Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

    doi: 10.1101/2023.03.22.533832

    Figure Lengend Snippet: (A) Still images from a lipid FRAP series of BODIPY-PI(3)P in GUVs in the presence of 100 µM Ca 2+ with and without 0.5 µM ANXA11 FL. Scale Bar - 5 µm. (B) PI(3)P fluorescence recovery rates of GUVs in the presence of 100 µM Ca 2+ co-incubated with either 0.5 µM ANXA11 FL or 0.5 µM ARD. The % fluorescence recovery by 5 sec is plotted alongside. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ns - not significant (p > 0.05), n=4. (C) A schematic illustrating the change in PK dye fluorescence emission as lipids transition from a disordered (red emission) to ordered (blue emission) state. (D) Fluorescence images of ATTO647 GUVs labelled with 5 µM PK dye to extract the relative lipid order (φ) of GUVs alone or with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or 0.5 µM ARD. Scale Bar - 5 µm. (E) Quantification of the relative lipid order (φ) of GUVs as shown in (D), including protein alone and Ca 2+ alone controls. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant, n=3 (30-82 GUVs). (F) A schematic of our AFM-IR setup which acquires nanoscale resolved chemical spectra of protein and lipid components. (G) An exemplary AFM-IR morphology map of GUV fragments bound to ANXA11. Green crosses indicate regions with a ‘lipid only’ signature. Red crosses indicate regions with ‘protein and lipid’ signatures. Scale bars - 2 µm. (H) A ratio of the infrared maps from protein:lipid (1655/1730 cm -1 ) spectroscopic signatures illustrates the heterogeneity of their spatial distribution at nanoscale. Scale bars - 2 µm. (I) A comparison of the average (Mean ± SD) AFM-IR spectra of GUV fragments bound to 0.5 µM ANXA11 FL and 0.5 µM ARD at 100 µM Ca 2+ . 450 spectra were collected from across 69 independent GUV fragments (12 lipid only, 31 lipid+FL, 26 lipid+ARD) across 5 experimental repeats. The SD in the ARD condition falls beneath the thickness of the line. (J) Second derivatives of the spectra (Mean ± SD) in the IR absorption of the C=O stretching region of lipids for GUV fragments alone or bound to either ANXA11 FL or ARD. (K) Quantification of the wavenumber of GUV fragments alone compared with GUV fragments bound to ANXA11 FL or ARD. The ∼1730 to ∼1738 cm -1 shift indicates a liquid-to-gel lipid phase transition. Mean ± SD and 25-75 th percentile (box). One-way ANOVA with Tukey’s multiple comparison, ***p < 0.001, ns - not significant (p > 0.05), n=5 (12-26 GUV fragments).

    Article Snippet: To test the effect of ANXA11 on lipid mobility, 20 μM GUVs in which the PI(3)P was replaced with fluorescent BODIPY-PI(3)P (Echelon) were incubated with or without 0.5 μM AF647-labelled ANXA11 FL for 15-30 min at 37 °C in 100 μM Ca 2+ as described above.

    Techniques: Fluorescence, Incubation, Sublimation

    Journal: Cell

    Article Title: Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics

    doi: 10.1016/j.cell.2022.10.018

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: BODIPY-TMR phosphatidylinositol 4,5-bisphosphate (BODIPY-PIP 2 ) (Echelon Biosciences) was dissolved to a stock concentration of 1 mM in 50 mM HEPES pH 7.4 and used at a final concentration of 4 nM in the assay.

    Techniques: Recombinant, Electrophoresis, Expressing, Plasmid Preparation, cAMP Assay, Clone Assay, Software, Flow Cytometry

    KEY RESOURCES TABLE

    Journal: Cell

    Article Title: Membrane phosphoinositides regulate GPCR-β-arrestin complex assembly and dynamics

    doi: 10.1016/j.cell.2022.10.018

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: BODIPY-TMR PI(4,5)P2 , Echelon Biosciences , Cat # C-45M6.

    Techniques: Recombinant, Electrophoresis, Expressing, Plasmid Preparation, cAMP Assay, Clone Assay, Software, Flow Cytometry

    Table 1

    Journal: Plant Signaling & Behavior

    Article Title: Phosphoinositide 5-Phosphate and Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes

    doi:

    Figure Lengend Snippet: Table 1

    Article Snippet: C-05R16 BODIPY-PI5P-tagged product from (Echelon Biosciences Inc., Salt Lake) was used to illustrate internalization and colocalization of exogenous PtdIns(5)P.

    Techniques:

    A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.

    Journal: PLoS ONE

    Article Title: Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P 2 -Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing

    doi: 10.1371/journal.pone.0103127

    Figure Lengend Snippet: A ) The addition of PI(4,5)P 2 increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P 2 or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + exoU or exoU-S142A , and analyzed with a cell retention assay using crystal violet staining. B ) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. C ) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P 2 . HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. D ) PI(4,5)P 2 dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P 2 for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.

    Article Snippet: Hydrolysis of PI(4,5)P 2 by rExoU and other phospholipases was examined by TLC analysis using BODIPY-FL-PI(4,5)P 2 , C 16 (Echelon) as a substrate.

    Techniques: Incubation, Infection, Staining, Colony-forming Unit Assay, Lactate Dehydrogenase Assay, Lysis

    A ) Enzymatic activity of rExoU on BODIPY-FL-PI(4,5)P 2 determined by TLC. The fluorescent substrate was incubated with 13.5 or 6.76 pmol rExoU in the presence of the cofactor ubiquitin (13.5 pmol), labeled as U + Ub. PI-PLC and honeybee PLA 2 , 13.5 pmol each, were also tested. The position of the non-hydrolyzed substrate is indicated with an arrow. The percentage hydrolysis of each enzyme, averaged from 3 independent experiments, is shown below the chromatograph. B ) Localization of ExoU-S142A in HeLa cells after infection in the presence or absence of the extracellular phospholipids. Infected cells were harvested, mechanically lysed, and fractionated. Fractions were analyzed by Western blot using an anti-ExoU monoclonal antibody. S: soluble fraction, M: membrane fraction. To achieve a linear detection range, 4-fold higher volumes of membrane fractions were loaded relative to soluble material. C ) The Western blot signal intensity of both unmodified and ubiquitinated ExoU-S142A was quantified. Membrane fraction (dark gray) and soluble fraction (light gray) are shown as a 100% stacked column chart with the mean ± SD from 3 independent experiments. D ) rExoU (13.5 pmol) binding to phospholipid (1600 pmol)-coated polystyrene plates detected by fluorescence microscopy. Magnification: 40x. E ) rExoU affinity to phospholipids evaluated by an ELISA-based solid-phase binding assay. Polystyrene plates were coated with the indicated amounts of phospholipids. The binding of 13.5 pmol rExoU was determined using an anti-ExoU antibody and a horseradish peroxidase-conjugated secondary antibody. Peroxidase activity on the substrate ABTS with H 2 O 2 was measured by absorbance at 405 nm. F ) The Kd of rExoU to PI(4,5)P 2 determined with the ELISA-based binding assay. Nonlinear regression analysis of the concentration of rExoU (nM) as a function of total binding is shown. The plot represents the mean ± SEM of duplicates from 2 independent experiments. G ) Binding of the PLCδ1-PH domain to PI(4,5)P 2 evaluated by nonlinear regression of the concentration of the PLCδ1-PH peptide (nM) as a function of total binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments. H ) Inhibitory effect of rExoU to PLCδ1-PH binding to PI(4,5)P 2 . rExoU (between 6.25 and 800 nM) was mixed into 50 nM of the PLCδ1-PH peptide for the solid-phase binding assay. IC 50 : the concentration of rExoU to displace 50% of PLCδ1-PH binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P 2 -Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing

    doi: 10.1371/journal.pone.0103127

    Figure Lengend Snippet: A ) Enzymatic activity of rExoU on BODIPY-FL-PI(4,5)P 2 determined by TLC. The fluorescent substrate was incubated with 13.5 or 6.76 pmol rExoU in the presence of the cofactor ubiquitin (13.5 pmol), labeled as U + Ub. PI-PLC and honeybee PLA 2 , 13.5 pmol each, were also tested. The position of the non-hydrolyzed substrate is indicated with an arrow. The percentage hydrolysis of each enzyme, averaged from 3 independent experiments, is shown below the chromatograph. B ) Localization of ExoU-S142A in HeLa cells after infection in the presence or absence of the extracellular phospholipids. Infected cells were harvested, mechanically lysed, and fractionated. Fractions were analyzed by Western blot using an anti-ExoU monoclonal antibody. S: soluble fraction, M: membrane fraction. To achieve a linear detection range, 4-fold higher volumes of membrane fractions were loaded relative to soluble material. C ) The Western blot signal intensity of both unmodified and ubiquitinated ExoU-S142A was quantified. Membrane fraction (dark gray) and soluble fraction (light gray) are shown as a 100% stacked column chart with the mean ± SD from 3 independent experiments. D ) rExoU (13.5 pmol) binding to phospholipid (1600 pmol)-coated polystyrene plates detected by fluorescence microscopy. Magnification: 40x. E ) rExoU affinity to phospholipids evaluated by an ELISA-based solid-phase binding assay. Polystyrene plates were coated with the indicated amounts of phospholipids. The binding of 13.5 pmol rExoU was determined using an anti-ExoU antibody and a horseradish peroxidase-conjugated secondary antibody. Peroxidase activity on the substrate ABTS with H 2 O 2 was measured by absorbance at 405 nm. F ) The Kd of rExoU to PI(4,5)P 2 determined with the ELISA-based binding assay. Nonlinear regression analysis of the concentration of rExoU (nM) as a function of total binding is shown. The plot represents the mean ± SEM of duplicates from 2 independent experiments. G ) Binding of the PLCδ1-PH domain to PI(4,5)P 2 evaluated by nonlinear regression of the concentration of the PLCδ1-PH peptide (nM) as a function of total binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments. H ) Inhibitory effect of rExoU to PLCδ1-PH binding to PI(4,5)P 2 . rExoU (between 6.25 and 800 nM) was mixed into 50 nM of the PLCδ1-PH peptide for the solid-phase binding assay. IC 50 : the concentration of rExoU to displace 50% of PLCδ1-PH binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments.

    Article Snippet: Hydrolysis of PI(4,5)P 2 by rExoU and other phospholipases was examined by TLC analysis using BODIPY-FL-PI(4,5)P 2 , C 16 (Echelon) as a substrate.

    Techniques: Activity Assay, Incubation, Labeling, Infection, Western Blot, Binding Assay, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Concentration Assay

    A ) After translocation into the host cytoplasm by T3SS, ExoU (U) is postulated to bind to PI(4,5)P 2 residing on the inner leaflet of the plasma membrane. The PLA 2 activity of ExoU is activated by a cofactor (Co), ubiquitin or ubiquitinated proteins. B ) ExoU hydrolyzes PI(4,5)P 2 and/or other phospholipid species (shown as Xs), leaving lyso-phospholipids, destabilizing the membrane. Reduction of PI(4,5)P 2 disrupts the anchoring and interaction between the focal adhesion, cytoskeletal structure, and plasma membrane, possibly through a cell signaling pathway. Cells round due to the weakened adhesion capability and actin filament depolymerization, leading to cytoskeletal collapse. The outer leaflet of the plasma membrane is intact at this stage. C ) At a middle stage of infection, the plasma membrane starts to bleb, facilitating further membrane damage. D ) During a late stage of infection, the outer leaflet of the plasma membrane is compromised, allowing the influx of an impermeable nucleic acid dye and staining of the nucleus (green), consequently large molecules (e.g. LDH) are released from lysed cells.

    Journal: PLoS ONE

    Article Title: Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P 2 -Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing

    doi: 10.1371/journal.pone.0103127

    Figure Lengend Snippet: A ) After translocation into the host cytoplasm by T3SS, ExoU (U) is postulated to bind to PI(4,5)P 2 residing on the inner leaflet of the plasma membrane. The PLA 2 activity of ExoU is activated by a cofactor (Co), ubiquitin or ubiquitinated proteins. B ) ExoU hydrolyzes PI(4,5)P 2 and/or other phospholipid species (shown as Xs), leaving lyso-phospholipids, destabilizing the membrane. Reduction of PI(4,5)P 2 disrupts the anchoring and interaction between the focal adhesion, cytoskeletal structure, and plasma membrane, possibly through a cell signaling pathway. Cells round due to the weakened adhesion capability and actin filament depolymerization, leading to cytoskeletal collapse. The outer leaflet of the plasma membrane is intact at this stage. C ) At a middle stage of infection, the plasma membrane starts to bleb, facilitating further membrane damage. D ) During a late stage of infection, the outer leaflet of the plasma membrane is compromised, allowing the influx of an impermeable nucleic acid dye and staining of the nucleus (green), consequently large molecules (e.g. LDH) are released from lysed cells.

    Article Snippet: Hydrolysis of PI(4,5)P 2 by rExoU and other phospholipases was examined by TLC analysis using BODIPY-FL-PI(4,5)P 2 , C 16 (Echelon) as a substrate.

    Techniques: Translocation Assay, Activity Assay, Infection, Staining