snap tag alexafluor 647  (New England Biolabs)


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    Structured Review

    New England Biolabs snap tag alexafluor 647
    Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and <t>SNAP-tagged</t> β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface <t>AF647-labeled</t> co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p
    Snap Tag Alexafluor 647, supplied by New England Biolabs, 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 "Complex Formation between VEGFR2 and the β2-Adrenoceptor"

    Article Title: Complex Formation between VEGFR2 and the β2-Adrenoceptor

    Journal: Cell Chemical Biology

    doi: 10.1016/j.chembiol.2019.02.014

    Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p
    Figure Legend Snippet: Effect of Agonist Stimulation on Receptor Oligomerization (A) Schematic of experimental setup to investigate the effect of isoprenaline or VEGF 165 a on receptor oligomerization measured using NanoBRET. (B) Visualization of VEGFR2/β 2 -adrenoceptor oligomers by NanoBRET using a luminescence LV200 Olympus microscope. HEK293 cells were transiently co-transfected to express NLuc-tagged-VEGFR2 and SNAP-tagged β 2 -adrenoceptors. Sequential images were captured from unlabeled (top panels) or SNAP-surface AF647-labeled co-transfected cells (bottom panels). Sequential images were acquired by capturing DAPI channel, displayed in the left panels (donor detection; using a 438/24 nm emission filter, 5 s exposure time), followed by CY5 channel, displayed in the right panels (BRET-excited acceptor, using a 647 long-pass filter, 30 s exposure time). Scale bar represents 20 μm. (C and D) HEK293 cells were transiently transfected with 0.05 μg/well NLuc-VEGFR2 and 0.10 μg/well SNAP-β 2 -AR and treated for 1 h at 37°C with increasing concentrations of (C) VEGF 165 a or (D) isoprenaline. Bar C corresponds to untreated (control) condition. Data are means ± SEM from five separate experiments, each performed in quadruplicate. **p

    Techniques Used: Microscopy, Transfection, Labeling, Bioluminescence Resonance Energy Transfer

    Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p
    Figure Legend Snippet: Influence of Agonists on the Cellular Location of Receptors and on Complex Formation between β 2 -Adrenoceptors and β-Arrestin2 (A) Confocal imaging (Zeiss LSM 710) of HEK293 cells transiently co-transfected with 0.25 μg/well HaloTag- VEGFR2 and 0.25 μg/well SNAP-β 2 -AR cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a ligands (30 min at 37°C). Data are representative of three individual experiments. Scale bar represents 20 μm. (B) Immunolabeling of early endosomes (anti-Rab 5 antibody labeling). HEK293 cells transiently co-transfected with 0.5 μg/well HaloTag-VEGFR2 (green) and 0.5 μg/well SNAP-β 2 -AR (red) cDNAs, under unstimulated conditions (vehicle) or after treatment with 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C). Cells were fixed using 3% paraformaldehyde/PBS, permeabilized using Triton X-100 (0.025% in PBS) and Rab 5 endosomal compartments labeled (cyan). Cells were imaged using a LSM880 confocal microscope (Zeiss). Data are representative of three individual experiments. Scale bar represents 10 μm. (C) Structured illumination microscopy (SIM) super-resolution images of HEK293 cells transiently co-transfected with HaloTag-VEGFR2 (green) and SNAP-β 2 -AR (red; 3 μg total cDNA). Cells were incubated with vehicle, 10 μM isoprenaline or 10 nM VEGF 165 a (30 min at 37°C) before fixation and mounting onto microscope slides. Coverslips were imaged using a Zeiss ELYRA PS.1 microscope. Areas of co-localized HaloTag-VEGFR2 and SNAP-β 2 -AR-labeled receptors are shown in yellow. Scale bar represents 10 μm. (D and E) Summary of Pearson's correlation coefficients (D) obtained following co-localization analysis of SIM images of circular regions of interest (ROI) in HEK293 cells co-expressing HaloTag-VEGFR2 and SNAP-β 2 -AR. ROI were placed on areas of fluorescence either at the plasma membrane or intracellular regions of SIM images of HEK293 cells co-expressing HaloTag-VEGFR2 (green; HaloTag AF488 membrane impermeant label) and SNAP-β 2 -AR (red; SNAP AF647 membrane impermeant label). TetraSpeck microspheres (0.1-μm spectral beads stained with four fluorophores: 365/430 nm [blue], 505/515 nm [green], 560/580 nm [orange], and 660/680 nm [red]) were included in each experiment to allow X/Y/Z channel alignment correction in image processing. The Fiji (ImageJ) analysis program CoLoc2 was applied to these ROI (six ROIs for spectral bead images and 12–15 ROIs for all other conditions) and Pearson's correlation coefficients obtained. Values were averaged across all ROI and are expressed as means ± SEM. A Pearson correlation coefficient value of +1 implies a perfect co-occurrence of both green (HaloTag-VEGFR2) and red (SNAP-β 2 -AR) fluorophores. *p

    Techniques Used: Imaging, Transfection, Immunolabeling, Antibody Labeling, Labeling, Microscopy, Incubation, Expressing, Fluorescence, Staining

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    New England Biolabs snap surface alexafluor647
    Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and <t>AlexaFluor647</t> conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.
    Snap Surface Alexafluor647, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Journal: bioRxiv

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    doi: 10.1101/2020.05.23.112607

    Figure Lengend Snippet: Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Article Snippet: This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day.

    Techniques: Transfection, Construct, Staining

    TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Journal: bioRxiv

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    doi: 10.1101/2020.05.23.112607

    Figure Lengend Snippet: TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Article Snippet: This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day.

    Techniques:

    EGF-dependent ROS-generation couples EGFR-phosphorylation to RPTPγ oxidation A. Reaction schematic of EGFR-dependent PTP-oxidation: Phosphorylated EGFR (red circles) activates PI3K, which results in the activation of Rac-GTPase and the cytosolic components of NOX-assembly like p40 phox , p47 phox and p67 phox . Recruitment of these components to the PM-based major NOX-unit and p22 phox subunit, aids the transfer of electrons from the cytosolic NADPH to extracellular oxygen (O 2 ) leading to the formation of superoxide anion (O 2 - ) that dismutates to hydrogen peroxide (H 2 O 2 ). Diffusion of H 2 O 2 through the PM causes the cysteine oxidation of the PM-vicinal PTPs, from thiol (SH) to sulfenic acid (SOH) state. B. Schematic of FLIM approach for the quantitative imaging of PTP-oxidation in live cells: Binding of DyTo (atto590 acceptor) to oxidized cysteines of PTP-mCitrine (donor) results in FRET between donor and acceptor reducing the excited state lifetime of the donor (τ DA ). Spatial invariance of τ DA and τ D enable the mapping of the fraction of oxidized PTP-mCitrine (a ox , local parameter) by global analysis. C. in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representativ confocal micrographs of RPTPγ-mCitrine in MCF7 WT cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (a ox , bottom row), upon 10’ stimulation with EGF-Alexa647 (0-160 ng/ml). Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy α L ) and corresponding EGF-Alexa647, or H 2 O 2 concentration in cells co-expressing EGFR-mTFP, RPTPγ-mCitrine or RPTPγ C1060S -mCitrine. Individual cells with mean±SD, N=3, n=13-15. ****p

    Journal: bioRxiv

    Article Title: RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

    doi: 10.1101/2022.06.01.494340

    Figure Lengend Snippet: EGF-dependent ROS-generation couples EGFR-phosphorylation to RPTPγ oxidation A. Reaction schematic of EGFR-dependent PTP-oxidation: Phosphorylated EGFR (red circles) activates PI3K, which results in the activation of Rac-GTPase and the cytosolic components of NOX-assembly like p40 phox , p47 phox and p67 phox . Recruitment of these components to the PM-based major NOX-unit and p22 phox subunit, aids the transfer of electrons from the cytosolic NADPH to extracellular oxygen (O 2 ) leading to the formation of superoxide anion (O 2 - ) that dismutates to hydrogen peroxide (H 2 O 2 ). Diffusion of H 2 O 2 through the PM causes the cysteine oxidation of the PM-vicinal PTPs, from thiol (SH) to sulfenic acid (SOH) state. B. Schematic of FLIM approach for the quantitative imaging of PTP-oxidation in live cells: Binding of DyTo (atto590 acceptor) to oxidized cysteines of PTP-mCitrine (donor) results in FRET between donor and acceptor reducing the excited state lifetime of the donor (τ DA ). Spatial invariance of τ DA and τ D enable the mapping of the fraction of oxidized PTP-mCitrine (a ox , local parameter) by global analysis. C. in cell EGF-dose response imaging for RPTPγ-mCitrine oxidation. Left panel: Representativ confocal micrographs of RPTPγ-mCitrine in MCF7 WT cells (top row) together with its oxidized fraction estimated using DyTo-FLIM (a ox , bottom row), upon 10’ stimulation with EGF-Alexa647 (0-160 ng/ml). Scale bar: 10 μm. Right panel: Quantification depicting the PM-proximal (orange) and PM-distal (blue) oxidized fractions as functions of receptor occupancy α L ) and corresponding EGF-Alexa647, or H 2 O 2 concentration in cells co-expressing EGFR-mTFP, RPTPγ-mCitrine or RPTPγ C1060S -mCitrine. Individual cells with mean±SD, N=3, n=13-15. ****p

    Article Snippet: Before the experiment, Snap-EGFR was labeled with 0.5 μM Snap-Surface Alexa647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60’.

    Techniques: Activation Assay, Diffusion-based Assay, Imaging, Binding Assay, Concentration Assay, Expressing

    RPTPγ is a suppressor of oncogenic EGFR promigratory signaling. A. Quantification of ectopic EGFR-mCitrine expression in WT (red), RPTPγ-KO (blue) and p22 phox -KO (green) MCF7 cells. B. Representative cell contour maps showing the temporal changes (color bar: time (min)) in the cell morphology for WT (upper row), RPTPγ-KO (middle row) and p22 phox -KO (bottom row) MCF7 cells, expressing PM-marker BFP-tkRas without (first column) or during 60’ EGF-Alexa647 stimulus: 1 ng/ml (second column); 160 ng/ml (third column). C. Quantification of EGF-stimulus induced morphodynamics at endogenous EGFR (~10 3 receptors/cell) in MCF7 cells (n= 9-20), integrated over time by the ratio of the perimeter of an equiareal circle to the actual perimeter of the cells (P circle /P cell ). First row: WT, second row: RPTPγ-KO, third row: p22phox-KO MCF7 cells. D. Same as (C) for MCF7 cells ectopically expressing EGFR-mCitrine (~2×10 5 receptors/cell). n=11-39. *p

    Journal: bioRxiv

    Article Title: RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

    doi: 10.1101/2022.06.01.494340

    Figure Lengend Snippet: RPTPγ is a suppressor of oncogenic EGFR promigratory signaling. A. Quantification of ectopic EGFR-mCitrine expression in WT (red), RPTPγ-KO (blue) and p22 phox -KO (green) MCF7 cells. B. Representative cell contour maps showing the temporal changes (color bar: time (min)) in the cell morphology for WT (upper row), RPTPγ-KO (middle row) and p22 phox -KO (bottom row) MCF7 cells, expressing PM-marker BFP-tkRas without (first column) or during 60’ EGF-Alexa647 stimulus: 1 ng/ml (second column); 160 ng/ml (third column). C. Quantification of EGF-stimulus induced morphodynamics at endogenous EGFR (~10 3 receptors/cell) in MCF7 cells (n= 9-20), integrated over time by the ratio of the perimeter of an equiareal circle to the actual perimeter of the cells (P circle /P cell ). First row: WT, second row: RPTPγ-KO, third row: p22phox-KO MCF7 cells. D. Same as (C) for MCF7 cells ectopically expressing EGFR-mCitrine (~2×10 5 receptors/cell). n=11-39. *p

    Article Snippet: Before the experiment, Snap-EGFR was labeled with 0.5 μM Snap-Surface Alexa647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60’.

    Techniques: Expressing, Marker

    RPTPγ-EGFR monomer interaction enables switch-like responses at low EGFR expression A. EGFR ( pY1068) , Erk ( pT202 and pY204) and Akt ( pS473) phosphorylation response in WT MCF7 cells as function of EGF concentration derived from Western blot analysis. mean±SD, N=2. B. Same as (A) comparing WT (red), to RPTPγ-KO (blue) MCF7 cells. Normalized phosphorylation versus EGF dose (ng/ml) together with corresponding EGF-receptor occupancy (α L ) is shown without, and upon 5’ stimulation with different doses of EGF-Alexa647. mean±SD, N=3. **p

    Journal: bioRxiv

    Article Title: RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

    doi: 10.1101/2022.06.01.494340

    Figure Lengend Snippet: RPTPγ-EGFR monomer interaction enables switch-like responses at low EGFR expression A. EGFR ( pY1068) , Erk ( pT202 and pY204) and Akt ( pS473) phosphorylation response in WT MCF7 cells as function of EGF concentration derived from Western blot analysis. mean±SD, N=2. B. Same as (A) comparing WT (red), to RPTPγ-KO (blue) MCF7 cells. Normalized phosphorylation versus EGF dose (ng/ml) together with corresponding EGF-receptor occupancy (α L ) is shown without, and upon 5’ stimulation with different doses of EGF-Alexa647. mean±SD, N=3. **p

    Article Snippet: Before the experiment, Snap-EGFR was labeled with 0.5 μM Snap-Surface Alexa647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60’.

    Techniques: Expressing, Concentration Assay, Derivative Assay, Western Blot

    In cell dose-response analysis reveals toggle switch dynamics for EGFR phosphorylation A. Quantitative imaging of EGFR phosphorylation: Binding of PTB-mCherry (acceptor) to phosphorylated EGFR-mCitrine (donor) induces FRET between donor and acceptor resulting in a reduced excited state lifetime (τ DA ) of the EGFR-mCitrine/ PTB-mCherry complex. Unphosphorylated EGFR-mCitrine exhibits a discrete fluorescence lifetime (τ D ) that is distinct from τ DA . The spatially invariant τ DA and τ D are shared global parameters that enable the mapping of the fraction of phosphorylated EGFR-mCitrine (α p , local parameter) within living cells by global analysis. B. Representative fluorescence micrographs of in cell dose-response imaging for EGFR phosphorylation after indicated increments of EGF-Alexa647 (0–320 ng/mL) at 1.5’ interval. First row: EGF-Alex647; Second row: EGFR-mCitrine; Third row: phosphorylated EGFR fraction (α p ); Fourth row: RPTPγ-mTFP; Scale bar: 10 μm. C. EGFR-mCitrine phosphorylation (α p ) plotted as a function of EGF-receptor occupancy (α L ) at the PM to incremental EGF-Alexa647 doses in WT (red), RPTPγ-KO (blue), RPTPγ-KO with RPTPγ-mTFP ectopic expression (yellow) and p22 phox -KO (green) MCF7cells D. same as (C) for WT (red), TCPTP-KO (blue) and TCPTP-KO with TCPTP-mTFP ectopic expression (yellow) MCF7cells. Solid lines: moving medians from single cell profiles; shaded bounds: median absolute deviations. N=3-4, n=12-14 E. EGFR-PTP network architecture depicting the state-transitions and regulatory interactions. Solid arrows: molecular state transitions (p: phosphorylation, I: inactive, A: active), dashed arrows: causal links. Kinetic constants, γ 1 , γ 2 : phosphatase rate constant of RPTPγ and TCPTP, k 1 : rate constant for PTP reactivation. ε 1 , ε 2 and ε 3 : (auto)-catalytic kinase rate constants for EGFR, ε 4 : rate constant for EGFR-dependent phosphatase inactivation. EGFR: EGFR monomer; EGFRp: phosphorylated EGFR monomer; EGF-EGFRp: liganded phosphorylated EGFR monomer, EGF-EGFR2: liganded EGFR dimer at 1:2 stoichiometry. F. Experimentally reconstructed 3D-bifurcation diagram showing the dependence of EGFR phosphorylation (α p ) on γ 1 .RPTPγ/EGFR and EGF-receptor occupancy (α L ). Bifurcation surface for RPTPγ-KO cells in which RPTPγ-mTFP was ectopically expressed together with EGFR-mCitrine. Yellow line: experimentally derived dose response trajectory. G. Same as (F) for TCPTP-KO cells in which TCPTP-mTFP was ectopically expressed together with EGFR-mCitrine. H. Same as (F) for RPTPγ-KO cells ectopically expressing EGFR-mCitrine. Red line: experimentally derived dose response trajectory of WT MCF7 cells ectopically expressing EGFR-mCitrine. Purple line: experimentally derived dose response trajectory of TCPTP-KO cells ectopically expressing EGFR-mCitrine. Blue line: experimentally derived dose response trajectory of RPTPγ-KO cells ectopically expressing EGFR-mCitrine. I. Fold changes in kinetic parameters relative to WT for (F). J. Fold changes in kinetic parameters relative to WT for (G).

    Journal: bioRxiv

    Article Title: RPTPγ is a redox-regulated suppressor of promigratory EGFR signaling

    doi: 10.1101/2022.06.01.494340

    Figure Lengend Snippet: In cell dose-response analysis reveals toggle switch dynamics for EGFR phosphorylation A. Quantitative imaging of EGFR phosphorylation: Binding of PTB-mCherry (acceptor) to phosphorylated EGFR-mCitrine (donor) induces FRET between donor and acceptor resulting in a reduced excited state lifetime (τ DA ) of the EGFR-mCitrine/ PTB-mCherry complex. Unphosphorylated EGFR-mCitrine exhibits a discrete fluorescence lifetime (τ D ) that is distinct from τ DA . The spatially invariant τ DA and τ D are shared global parameters that enable the mapping of the fraction of phosphorylated EGFR-mCitrine (α p , local parameter) within living cells by global analysis. B. Representative fluorescence micrographs of in cell dose-response imaging for EGFR phosphorylation after indicated increments of EGF-Alexa647 (0–320 ng/mL) at 1.5’ interval. First row: EGF-Alex647; Second row: EGFR-mCitrine; Third row: phosphorylated EGFR fraction (α p ); Fourth row: RPTPγ-mTFP; Scale bar: 10 μm. C. EGFR-mCitrine phosphorylation (α p ) plotted as a function of EGF-receptor occupancy (α L ) at the PM to incremental EGF-Alexa647 doses in WT (red), RPTPγ-KO (blue), RPTPγ-KO with RPTPγ-mTFP ectopic expression (yellow) and p22 phox -KO (green) MCF7cells D. same as (C) for WT (red), TCPTP-KO (blue) and TCPTP-KO with TCPTP-mTFP ectopic expression (yellow) MCF7cells. Solid lines: moving medians from single cell profiles; shaded bounds: median absolute deviations. N=3-4, n=12-14 E. EGFR-PTP network architecture depicting the state-transitions and regulatory interactions. Solid arrows: molecular state transitions (p: phosphorylation, I: inactive, A: active), dashed arrows: causal links. Kinetic constants, γ 1 , γ 2 : phosphatase rate constant of RPTPγ and TCPTP, k 1 : rate constant for PTP reactivation. ε 1 , ε 2 and ε 3 : (auto)-catalytic kinase rate constants for EGFR, ε 4 : rate constant for EGFR-dependent phosphatase inactivation. EGFR: EGFR monomer; EGFRp: phosphorylated EGFR monomer; EGF-EGFRp: liganded phosphorylated EGFR monomer, EGF-EGFR2: liganded EGFR dimer at 1:2 stoichiometry. F. Experimentally reconstructed 3D-bifurcation diagram showing the dependence of EGFR phosphorylation (α p ) on γ 1 .RPTPγ/EGFR and EGF-receptor occupancy (α L ). Bifurcation surface for RPTPγ-KO cells in which RPTPγ-mTFP was ectopically expressed together with EGFR-mCitrine. Yellow line: experimentally derived dose response trajectory. G. Same as (F) for TCPTP-KO cells in which TCPTP-mTFP was ectopically expressed together with EGFR-mCitrine. H. Same as (F) for RPTPγ-KO cells ectopically expressing EGFR-mCitrine. Red line: experimentally derived dose response trajectory of WT MCF7 cells ectopically expressing EGFR-mCitrine. Purple line: experimentally derived dose response trajectory of TCPTP-KO cells ectopically expressing EGFR-mCitrine. Blue line: experimentally derived dose response trajectory of RPTPγ-KO cells ectopically expressing EGFR-mCitrine. I. Fold changes in kinetic parameters relative to WT for (F). J. Fold changes in kinetic parameters relative to WT for (G).

    Article Snippet: Before the experiment, Snap-EGFR was labeled with 0.5 μM Snap-Surface Alexa647 (New England Biolabs GmbH, Frankfurt, Germany) for at least 60’.

    Techniques: Imaging, Binding Assay, Fluorescence, Expressing, Derivative Assay

    Cryo-arrest of a two-component molecular signaling system. ( A ) Dual-color CLSM images of R-PTP-γ-mCitrine (magenta) and Alexa647-Snap-EGFR (green) in MCF7 cells during EGF (100 ng/ml) stimulation before (at indicated time, 3.75-s scan time) and during cryo-arrest (10 frames, 37 s). Right: CLSM of a larger area during cryo-arrest. ( B ) Motional blur measured by CLSM of Alexa647-Snap-EGFR–labeled endosomes at room temperature (top) and under cryo-arrest (bottom). Left images: individual frame (2.5 × 2.5 μm); right image: sum of 10 frames. Graphs: corresponding cumulative background corrected line profiles color coded by frame number. ( C ) Motional blur measured by pattern similarity between frames. Lines: coefficient of determination ( r 2 ) of individual regions containing endosomes at room temperature (orange) and under cryo-arrest (blue); mean ± SD (black; n = 6). ( D ) Alexa647 photobleaching at room temperature (orange; n = 5) and under cryo-arrest (blue; n = 7) during 10 confocal scans. λ: photobleaching rates (mean ± SD). ( E ) Dual-color SRRF reconstruction from 100 wide-field frames of cryo-arrested cell depicted in (A) (left) and a cryo-arrested unstimulated cell (right). Bottom: magnifications of boxed areas; dotted lines: plasma membrane area. ( F ) Manders’ colocalization coefficients for Alexa647-Snap-EGFR/R-PTP-γ-mCitrine from SRRF reconstructions under cryo-arrest in endosomes (left) or plasma membrane area (right) for unstimulated (−EGF; n = 18) and 15-min EGF-stimulated cells (+EGF; n = 15); P : Student’s t test. Scale bars: 10 μm.

    Journal: Science Advances

    Article Title: Ultrarapid cryo-arrest of living cells on a microscope enables multiscale imaging of out-of-equilibrium molecular patterns

    doi: 10.1126/sciadv.abk0882

    Figure Lengend Snippet: Cryo-arrest of a two-component molecular signaling system. ( A ) Dual-color CLSM images of R-PTP-γ-mCitrine (magenta) and Alexa647-Snap-EGFR (green) in MCF7 cells during EGF (100 ng/ml) stimulation before (at indicated time, 3.75-s scan time) and during cryo-arrest (10 frames, 37 s). Right: CLSM of a larger area during cryo-arrest. ( B ) Motional blur measured by CLSM of Alexa647-Snap-EGFR–labeled endosomes at room temperature (top) and under cryo-arrest (bottom). Left images: individual frame (2.5 × 2.5 μm); right image: sum of 10 frames. Graphs: corresponding cumulative background corrected line profiles color coded by frame number. ( C ) Motional blur measured by pattern similarity between frames. Lines: coefficient of determination ( r 2 ) of individual regions containing endosomes at room temperature (orange) and under cryo-arrest (blue); mean ± SD (black; n = 6). ( D ) Alexa647 photobleaching at room temperature (orange; n = 5) and under cryo-arrest (blue; n = 7) during 10 confocal scans. λ: photobleaching rates (mean ± SD). ( E ) Dual-color SRRF reconstruction from 100 wide-field frames of cryo-arrested cell depicted in (A) (left) and a cryo-arrested unstimulated cell (right). Bottom: magnifications of boxed areas; dotted lines: plasma membrane area. ( F ) Manders’ colocalization coefficients for Alexa647-Snap-EGFR/R-PTP-γ-mCitrine from SRRF reconstructions under cryo-arrest in endosomes (left) or plasma membrane area (right) for unstimulated (−EGF; n = 18) and 15-min EGF-stimulated cells (+EGF; n = 15); P : Student’s t test. Scale bars: 10 μm.

    Article Snippet: The cells were labeled with Snap-Surface Alexa647, and it was microscopically confirmed that > 99% of these cells also express SNAP-EGFR.

    Techniques: Confocal Laser Scanning Microscopy, Labeling

    STED nanoscopy of a dynamic receptor system under cryo-arrest. ( A ) CLSM and STED nanoscopy of Alexa647-Snap-EGFR in chemically fixed (rows 1 to 3), live at room temperature (fourth row), and cryo-arrested (bottom row) MCF7 cells. CLSM of cells (first column; pixel size: 100 nm); detailed (pixel size: 40 nm) CLSM (second column) and STED (third column) scan of boxed area with indicated laser irradiation intensities. Fourth column: overlay, CLSM (magenta), and STED (green). Live: single scan; cryo-arrest: sum of 10 scans. Graphs: intensity profiles along the dotted lines depicted in the CLSM/STED overlay images (STED: color-coded to lines; CLSM: magenta). ( B ) Alexa647 photobleaching during STED scans at room temperature (rt) or under cryo-arrest (cryo) at the indicated depletion laser powers. λ: decay rates determined by exponential fits (mean ± SD, N = 5). ( C ) STED spatial frequency spectrum normalized by corresponding CLSM spectrum for different regions containing vesicles (cryo: n = 11; chemical fixation: n = 8; rt. live: n = 6); Horizontal lines: frequency range where STED information content is significantly higher than CLSM (Student’s t test); vertical dashed line: digital resolution. ( D ) Top row: 3D-CLSM of a cryo-arrested MCF7 cell. Lower rows: detailed high-resolution scans (5 × 5 μm; pixel size: 40 nm) of the indicated regions in the top row: CLSM (cyan), STED (gray), and merge (magenta and green).

    Journal: Science Advances

    Article Title: Ultrarapid cryo-arrest of living cells on a microscope enables multiscale imaging of out-of-equilibrium molecular patterns

    doi: 10.1126/sciadv.abk0882

    Figure Lengend Snippet: STED nanoscopy of a dynamic receptor system under cryo-arrest. ( A ) CLSM and STED nanoscopy of Alexa647-Snap-EGFR in chemically fixed (rows 1 to 3), live at room temperature (fourth row), and cryo-arrested (bottom row) MCF7 cells. CLSM of cells (first column; pixel size: 100 nm); detailed (pixel size: 40 nm) CLSM (second column) and STED (third column) scan of boxed area with indicated laser irradiation intensities. Fourth column: overlay, CLSM (magenta), and STED (green). Live: single scan; cryo-arrest: sum of 10 scans. Graphs: intensity profiles along the dotted lines depicted in the CLSM/STED overlay images (STED: color-coded to lines; CLSM: magenta). ( B ) Alexa647 photobleaching during STED scans at room temperature (rt) or under cryo-arrest (cryo) at the indicated depletion laser powers. λ: decay rates determined by exponential fits (mean ± SD, N = 5). ( C ) STED spatial frequency spectrum normalized by corresponding CLSM spectrum for different regions containing vesicles (cryo: n = 11; chemical fixation: n = 8; rt. live: n = 6); Horizontal lines: frequency range where STED information content is significantly higher than CLSM (Student’s t test); vertical dashed line: digital resolution. ( D ) Top row: 3D-CLSM of a cryo-arrested MCF7 cell. Lower rows: detailed high-resolution scans (5 × 5 μm; pixel size: 40 nm) of the indicated regions in the top row: CLSM (cyan), STED (gray), and merge (magenta and green).

    Article Snippet: The cells were labeled with Snap-Surface Alexa647, and it was microscopically confirmed that > 99% of these cells also express SNAP-EGFR.

    Techniques: Confocal Laser Scanning Microscopy, Irradiation

    Additional Representative Fluorescence and FRET Traces Using AlexaFluor647-Labeled Cas10 and Cy3-Labeled WT RNA Idealized FRET states from hidden-Markov-modeling (HMM) analysis are overlaid in orange.

    Journal: bioRxiv

    Article Title: Dynamics of Cas10 Govern Discrimination between Self and Nonself in Type III CRISPR-Cas Immunity

    doi: 10.1101/369744

    Figure Lengend Snippet: Additional Representative Fluorescence and FRET Traces Using AlexaFluor647-Labeled Cas10 and Cy3-Labeled WT RNA Idealized FRET states from hidden-Markov-modeling (HMM) analysis are overlaid in orange.

    Article Snippet: Protein labeling SNAP-tagged Cas10-Csm protein complexes were labeled at a concentration of 5 μM with 10 μM SNAP-Surface AlexaFluor647 (New England Biolabs) in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM DTT.

    Techniques: Fluorescence, Labeling