topfluor tmr ps  (Avanti Polar)

Journal: EMBO Reports

Article Title: Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

doi: 10.15252/embr.202051709

Figure Lengend Snippet: A Representative confocal images of fluorescently labeled (TopFluor® TMR‐PS) GUVs with varying lipid compositions (red) following incubation with eVP40‐Alexa88 (green). Colocalization between eVP40 and PS within GUVs was indicated by plot profile analyses of fluorescence signals between TopFluor® TMR‐PS (red dotted line) and eVP40‐Alexa488 (green solid line) performed at indicated open yellow lines in a and shown in (B–D). B Plot profile analysis of eVP40‐Alexa488 and control GUVs (DPPC:Cholesterol:0.2 mol% TopFluor® TMR‐PS). C Plot profile analysis of eVP40‐Alexa488 and PS GUVs (DPPC:Cholesterol:0.2 mol% TopFluor® TMR‐PS:DPPS). * indicates overlap in fluorescence signals. D Plot profile analysis of eVP40‐Alexa488 and PS + PI(4,5)P 2 GUVs (DPPC:Cholesterol:0.2 mol% TopFluor® TMR‐PS:DPPS:PI(4,5)P 2 ). E Protein enrichment analysis (eVP40‐Alexa488) measured as the fluorescence of eVP40 (Alexa488 labeled) at the GUV membrane divided by total fluorescence. **** P < 0.0001, n = 3 biological replicates. Data information: DPPC, dipalmitoyl‐phosphatidylcholine; DPPS, dipalmitoyl‐phosphatidylserine; PS, phosphatidylserine.

Article Snippet: POPC (#850457), DPPC (#850355), Chol (#700000), DPPS (#840037), POPE (#850757), POPS (#840034), Brain PI(4,5)P 2 (#840046), and TopFluor® TMR‐PS (#810242) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and stored in chloroform and/or methanol at −20°C until use.

Techniques: Labeling, Incubation, Fluorescence, Control, Protein Enrichment, Membrane

Journal: EMBO Reports

Article Title: Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

doi: 10.15252/embr.202051709

Figure Lengend Snippet: A Representative confocal images from live cell imaging of HEK293 expressing various GFP‐fused proteins (GFP; green) following supplementation with TopFluor® TMR‐PS (red). Solid white lines indicate where plot profile analysis was performed; scale bar = 10 μm. B, C Validation of ability to detect exogenously added fluorescently labeled PS within the inner leaflet of the plasma membrane of cells (B) Plot profile analysis of HEK293 cells expressing cytosolic GFP. (C) Plot profile analysis of HEK293 cells expressing the PS sensor GFP‐LactC2. D, E Investigation of functionally distinct eVP40 proteins ability to bind to fluorescently labeled PS within the inner leaflet of the plasma membrane in living cells. (D) Plot profile analysis of HEK293 cells expressing GFP‐WT‐eVP40. (E) Plot profile analysis of HEK293 cells expressing GFP‐K224A‐eVP40 (PS‐binding residue mutant). F Plot profile analysis of HEK293 cells expressing GFP‐WE/A‐eVP40 (oligomerization‐deficient mutant). Data information: (B–F) TopFluor® TMR‐PS fluorescence signal intensity (red dotted line) and GFP fluorescence signal intensity (green solid line).

Article Snippet: POPC (#850457), DPPC (#850355), Chol (#700000), DPPS (#840037), POPE (#850757), POPS (#840034), Brain PI(4,5)P 2 (#840046), and TopFluor® TMR‐PS (#810242) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and stored in chloroform and/or methanol at −20°C until use.

Techniques: Live Cell Imaging, Expressing, Biomarker Discovery, Labeling, Clinical Proteomics, Membrane, Binding Assay, Residue, Mutagenesis, Fluorescence

Journal: EMBO Reports

Article Title: Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

doi: 10.15252/embr.202051709

Figure Lengend Snippet: A Representative 3D reconstructed confocal images of immobilized GUVs (DPPC:Cholesterol:DPPS:PI(4,5)P 2 :TopFluor® TMR‐PS(red)). Left panel: GUVs incubated without eVP40‐Alexa488. Right three panels: GUVs incubated with 1.25 μM eVP40‐Alexa488 (green). Scale bar = 5 μm. B Index of correlation (Mander's coefficient) between TopFluor® TMR‐PS and eVP40‐Alexa488 of different GUVs compositions incubated with 1.25 μM eVP40‐Alexa488. Values are reported as mean ± s.d. A one‐way ANOVA with multiple comparisons was performed; n = 3 for 3 technical replicates **** P < 0.0001. C % TopFluor® TMR‐PS quenching by eVP40 using GUVs (DPPC:Cholesterol:TopFluor®TMR‐PS + increasing mol% of PS), 2.5% PI(4,5)P 2 was added to GUVs with 60% PS. Fluorescence spectra were recorded (Ex: 547 nm; Em: 550–600 nm); n = 2 technical replicates. D Representative confocal images of HEK293 cells expressing various GFP‐fused proteins (green) and supplemented with TopFluor® TMR‐PS (red); scale bar = 10 μm. Yellow arrows indicate high intensity PS fluorescent regions. E %PM with PS clusters = area of high intensity fluorescent PS clusters over total PM area from images in panel D. Black bars are control proteins and blue bars are eVP40 proteins. Values are reported as mean ± s.d.; N > 18 cells per biological replicate, n = 3 for 3 biological replicates; A one‐way ANOVA was performed with multiple comparisons compared with the control GFP %PS clustering (*** P = 0.0007, ** P = 0.004). Data information: DPPC, dipalmitoyl‐phosphatidylcholine; DPPS, dipalmitoyl‐phosphatidylserine; GUVs, giant unilamellar vesicles; PM, plasma membrane; PS: phosphatidylserine.

Article Snippet: POPC (#850457), DPPC (#850355), Chol (#700000), DPPS (#840037), POPE (#850757), POPS (#840034), Brain PI(4,5)P 2 (#840046), and TopFluor® TMR‐PS (#810242) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and stored in chloroform and/or methanol at −20°C until use.

Techniques: Incubation, Fluorescence, Expressing, Control, Clinical Proteomics, Membrane

Journal: EMBO Reports

Article Title: Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding

doi: 10.15252/embr.202051709

Figure Lengend Snippet: A Representative images of the step‐wise image analysis workflow of quantifying PS clustering in living HEK293 cells expressing GFP‐fused proteins using a custom ImageJ macro. Scale bar = 10 μm. B Representative images from live cell imaging experiments of HEK293 cells expressing control GFP‐fused proteins specific for the plasma membrane (GPI), and specific lipids, PS (LactC2) and PI(4,5)P 2 (PLCδ‐PH) following supplementation with TopFluor® TMR‐PS (red, middle panel). Scale bar = 10 μm.

Article Snippet: POPC (#850457), DPPC (#850355), Chol (#700000), DPPS (#840037), POPE (#850757), POPS (#840034), Brain PI(4,5)P 2 (#840046), and TopFluor® TMR‐PS (#810242) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and stored in chloroform and/or methanol at −20°C until use.

Techniques: Expressing, Live Cell Imaging, Control, Clinical Proteomics, Membrane