alexa647  (New England Biolabs)


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    Structured Review

    New England Biolabs alexa647
    Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP <t>(BC-Alexa647).</t> Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.
    Alexa647, 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 "Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance"

    Article Title: Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance

    Journal: Nature Communications

    doi: 10.1038/ncomms13334

    Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP (BC-Alexa647). Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.
    Figure Legend Snippet: Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP (BC-Alexa647). Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.

    Techniques Used: Immunofluorescence, Expressing, Cross-linking Immunoprecipitation, Staining, Fluorescence, HAC Assay, Transfection, Construct, Synthesized

    Related Articles

    Incubation:

    Article Title: Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility
    Article Snippet: .. To generate a fluorescently labelled SNAP‐BicD2‐N (referred to as Alexa647‐BicD2‐N), the His6‐SNAP‐BicD2‐N was incubated overnight at 4°C with an equimolar concentration of SNAP‐Surface Alexa Fluor 647 (New England Biolabs) during the TEV protease cleavage reaction. .. The labelled protein was then passed through HiPrep 26/60 desalting columns (GE Healthcare) to remove the unreacted dye.

    Concentration Assay:

    Article Title: Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility
    Article Snippet: .. To generate a fluorescently labelled SNAP‐BicD2‐N (referred to as Alexa647‐BicD2‐N), the His6‐SNAP‐BicD2‐N was incubated overnight at 4°C with an equimolar concentration of SNAP‐Surface Alexa Fluor 647 (New England Biolabs) during the TEV protease cleavage reaction. .. The labelled protein was then passed through HiPrep 26/60 desalting columns (GE Healthcare) to remove the unreacted dye.

    Mass Spectrometry:

    Article Title: The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
    Article Snippet: The tubulin containing fractions were pooled, concentrated (Vivaspin 30,000 MWCO, Sartorius) to above 3.5 mg/ml, ultracentrifuged (278,088 g , 10 min, 4°C), aliquoted, snap frozen, and stored in liquid nitrogen until use. .. Mass spectroscopy demonstrated that the purified human tubulin was free of insect cell tubulin ( ). mGFP-EB3 and SNAP-EB3 were purified and SNAP-EB3 was labeled with SNAP-Surface-AlexaFluor647 (NEB) as described (referred to as Alexa647-EB3 throughout the manuscript) ( ). .. Biotinylated monomeric Drosophila melanogaster kinesin-1 rigor mutant (Kin1rigor ) was purified as described previously ( ).

    Purification:

    Article Title: The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
    Article Snippet: The tubulin containing fractions were pooled, concentrated (Vivaspin 30,000 MWCO, Sartorius) to above 3.5 mg/ml, ultracentrifuged (278,088 g , 10 min, 4°C), aliquoted, snap frozen, and stored in liquid nitrogen until use. .. Mass spectroscopy demonstrated that the purified human tubulin was free of insect cell tubulin ( ). mGFP-EB3 and SNAP-EB3 were purified and SNAP-EB3 was labeled with SNAP-Surface-AlexaFluor647 (NEB) as described (referred to as Alexa647-EB3 throughout the manuscript) ( ). .. Biotinylated monomeric Drosophila melanogaster kinesin-1 rigor mutant (Kin1rigor ) was purified as described previously ( ).

    Article Title: Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes
    Article Snippet: Labeling efficiency was measured using a NanoDrop2000C (Thermo Scientific) and the labeled protein was stored at −80 °C. .. Purified SNAP-tag-αSNAP was diluted to 5 μM and labeled with 10 μM benzylguanine (BG)-Alexa647 dyes (New England Biolabs) for 2 h at room temperature or overnight at 4 °C. ..

    Labeling:

    Article Title: The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
    Article Snippet: The tubulin containing fractions were pooled, concentrated (Vivaspin 30,000 MWCO, Sartorius) to above 3.5 mg/ml, ultracentrifuged (278,088 g , 10 min, 4°C), aliquoted, snap frozen, and stored in liquid nitrogen until use. .. Mass spectroscopy demonstrated that the purified human tubulin was free of insect cell tubulin ( ). mGFP-EB3 and SNAP-EB3 were purified and SNAP-EB3 was labeled with SNAP-Surface-AlexaFluor647 (NEB) as described (referred to as Alexa647-EB3 throughout the manuscript) ( ). .. Biotinylated monomeric Drosophila melanogaster kinesin-1 rigor mutant (Kin1rigor ) was purified as described previously ( ).

    Article Title: Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization
    Article Snippet: Purified BicD2N and Hook3 was used to isolate DDB complexes from rat brain cytosol as previously described ( ). .. DDB complexes were labeled with in a ~4:1 ratio of dye:SNAPf-tagged protein at 2 μM SNAP-TMR, SNAP-Alexa647, or SNAP-Alexa488 dye (NEB) during the isolation procedure, and were frozen in small aliquots and stored at −80°C. .. The stoichiometry of labeling and protein concentration was assessed using a Nanodrop One (ThermoFisher) and comparing the absorbance of total protein at 280nm to the absorbance at the SNAP-dye wavelength.

    Article Title: Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes
    Article Snippet: Labeling efficiency was measured using a NanoDrop2000C (Thermo Scientific) and the labeled protein was stored at −80 °C. .. Purified SNAP-tag-αSNAP was diluted to 5 μM and labeled with 10 μM benzylguanine (BG)-Alexa647 dyes (New England Biolabs) for 2 h at room temperature or overnight at 4 °C. ..

    Article Title: Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy
    Article Snippet: Cellular conditions The following cellular conditions were used: 1) WTSAG , WT cell line treated with 100 nM SMO agonist (SAG) for 4 h; 2) W T S A G c b , WT cell line treated with 100 nM SAG for 4 h, then treated with 10 μM ciliobrevin D (cb ) for 1 h; 3) IFT25 NoAg , IFT25 knockout cell line not treated with any agonist at all; 4) IFT25 SAG , IFT25 knockout cell line treated with 100 nM SAG for 4 h. .. Sample preparation for 3D SR microscopy MEF cells are first labeled with Benzylguanine-Alexa647 (S9136S; NEB, Ipswich, MA), then fixed with 4% paraformaldehyde, and finally treated with a quenching solution of 10 mM NH4 Cl. ..

    Isolation:

    Article Title: Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization
    Article Snippet: Purified BicD2N and Hook3 was used to isolate DDB complexes from rat brain cytosol as previously described ( ). .. DDB complexes were labeled with in a ~4:1 ratio of dye:SNAPf-tagged protein at 2 μM SNAP-TMR, SNAP-Alexa647, or SNAP-Alexa488 dye (NEB) during the isolation procedure, and were frozen in small aliquots and stored at −80°C. .. The stoichiometry of labeling and protein concentration was assessed using a Nanodrop One (ThermoFisher) and comparing the absorbance of total protein at 280nm to the absorbance at the SNAP-dye wavelength.

    Cross-linking Immunoprecipitation:

    Article Title: Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance
    Article Snippet: The tetR-SNAP fusion proteins were detected by incubating the cells with either TMR-Star or SNAP-Cell Sir-647 (NEB) 30 min before fixation. .. CLIP-H3.3 was detected with benzylcytosine labelled with Alexa647 after fixation (NEB). .. Preparation and staining of unfixed metaphase chromosomes was performed as previously described .

    Sample Prep:

    Article Title: Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy
    Article Snippet: Cellular conditions The following cellular conditions were used: 1) WTSAG , WT cell line treated with 100 nM SMO agonist (SAG) for 4 h; 2) W T S A G c b , WT cell line treated with 100 nM SAG for 4 h, then treated with 10 μM ciliobrevin D (cb ) for 1 h; 3) IFT25 NoAg , IFT25 knockout cell line not treated with any agonist at all; 4) IFT25 SAG , IFT25 knockout cell line treated with 100 nM SAG for 4 h. .. Sample preparation for 3D SR microscopy MEF cells are first labeled with Benzylguanine-Alexa647 (S9136S; NEB, Ipswich, MA), then fixed with 4% paraformaldehyde, and finally treated with a quenching solution of 10 mM NH4 Cl. ..

    Microscopy:

    Article Title: Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy
    Article Snippet: Cellular conditions The following cellular conditions were used: 1) WTSAG , WT cell line treated with 100 nM SMO agonist (SAG) for 4 h; 2) W T S A G c b , WT cell line treated with 100 nM SAG for 4 h, then treated with 10 μM ciliobrevin D (cb ) for 1 h; 3) IFT25 NoAg , IFT25 knockout cell line not treated with any agonist at all; 4) IFT25 SAG , IFT25 knockout cell line treated with 100 nM SAG for 4 h. .. Sample preparation for 3D SR microscopy MEF cells are first labeled with Benzylguanine-Alexa647 (S9136S; NEB, Ipswich, MA), then fixed with 4% paraformaldehyde, and finally treated with a quenching solution of 10 mM NH4 Cl. ..

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    New England Biolabs alexa 647 dye
    CAMSAP CKKs protect MT minus ends from MCAK-induced depolymerization via steric inhibition. ( a ) A MT tubulin dimer-pair bound to CAMSAP3-CKK (green) is shown with the expected position of an MD of human MCAK (in complex with ADP; PDB ID 4UBF) by alignment with MT-bound kinesin-1 47  in Chimera 48 . b ) Kymographs of MT depolymerization assay with GMPCPP-stabilized MTs (blue) and GFP-MCAK (red). MT polarity was determined based on the movement of the SNAP-Alexa647-tagged plus-end directed motor kinesin-1 KIF5B (green, residues 1–560). Scale bars: horizontal, 1 μm; vertical, upper panel, 1 sec, lower panels, 1 min. c ) Kymographs of MT depolymerization assays with GMPCPP-stabilized MTs (blue), GFP-MCAK (red) and different concentrations of SNAP-Alexa 647 CAMSAP1 mini  or CKK (green). Scale bars: horizontal, 1 μm; vertical, 1 min. In panels (b) and (c), MT minus (−) ends are shown on the left and plus (+) ends on the right. d-e ) Quantification of MT depolymerization rate, MCAK intensity and CAMSAP1 intensity at different concentrations of CAMSAP1 mini  (d) or CKK (e). n ranged from 17 to 31 MTs; individual data points are provided in  Supplementary Table 2 . Data represent mean ± SD. See also  Supplementary Table 2 .
    Alexa 647 Dye, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs alexa647
    Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP <t>(BC-Alexa647).</t> Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.
    Alexa647, 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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    Average 86 stars, based on 1 article reviews
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    86
    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 SNAP-tagged 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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    Image Search Results


    CAMSAP CKKs protect MT minus ends from MCAK-induced depolymerization via steric inhibition. ( a ) A MT tubulin dimer-pair bound to CAMSAP3-CKK (green) is shown with the expected position of an MD of human MCAK (in complex with ADP; PDB ID 4UBF) by alignment with MT-bound kinesin-1 47  in Chimera 48 . b ) Kymographs of MT depolymerization assay with GMPCPP-stabilized MTs (blue) and GFP-MCAK (red). MT polarity was determined based on the movement of the SNAP-Alexa647-tagged plus-end directed motor kinesin-1 KIF5B (green, residues 1–560). Scale bars: horizontal, 1 μm; vertical, upper panel, 1 sec, lower panels, 1 min. c ) Kymographs of MT depolymerization assays with GMPCPP-stabilized MTs (blue), GFP-MCAK (red) and different concentrations of SNAP-Alexa 647 CAMSAP1 mini  or CKK (green). Scale bars: horizontal, 1 μm; vertical, 1 min. In panels (b) and (c), MT minus (−) ends are shown on the left and plus (+) ends on the right. d-e ) Quantification of MT depolymerization rate, MCAK intensity and CAMSAP1 intensity at different concentrations of CAMSAP1 mini  (d) or CKK (e). n ranged from 17 to 31 MTs; individual data points are provided in  Supplementary Table 2 . Data represent mean ± SD. See also  Supplementary Table 2 .

    Journal: Nature structural & molecular biology

    Article Title: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

    doi: 10.1038/nsmb.3483

    Figure Lengend Snippet: CAMSAP CKKs protect MT minus ends from MCAK-induced depolymerization via steric inhibition. ( a ) A MT tubulin dimer-pair bound to CAMSAP3-CKK (green) is shown with the expected position of an MD of human MCAK (in complex with ADP; PDB ID 4UBF) by alignment with MT-bound kinesin-1 47 in Chimera 48 . b ) Kymographs of MT depolymerization assay with GMPCPP-stabilized MTs (blue) and GFP-MCAK (red). MT polarity was determined based on the movement of the SNAP-Alexa647-tagged plus-end directed motor kinesin-1 KIF5B (green, residues 1–560). Scale bars: horizontal, 1 μm; vertical, upper panel, 1 sec, lower panels, 1 min. c ) Kymographs of MT depolymerization assays with GMPCPP-stabilized MTs (blue), GFP-MCAK (red) and different concentrations of SNAP-Alexa 647 CAMSAP1 mini or CKK (green). Scale bars: horizontal, 1 μm; vertical, 1 min. In panels (b) and (c), MT minus (−) ends are shown on the left and plus (+) ends on the right. d-e ) Quantification of MT depolymerization rate, MCAK intensity and CAMSAP1 intensity at different concentrations of CAMSAP1 mini (d) or CKK (e). n ranged from 17 to 31 MTs; individual data points are provided in Supplementary Table 2 . Data represent mean ± SD. See also Supplementary Table 2 .

    Article Snippet: To label SNAP tagged proteins with Alexa-647 dye (NEB), 20–40 μM dye was incubated with proteins on beads for 1 hr between the washing and elution steps.

    Techniques: Inhibition, Size-exclusion Chromatography

    Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Journal: Journal of structural biology

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    doi: 10.1016/j.jsb.2014.03.018

    Figure Lengend Snippet: Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Article Snippet: For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used.

    Techniques: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Labeling

    ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Journal: Journal of structural biology

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    doi: 10.1016/j.jsb.2014.03.018

    Figure Lengend Snippet: ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Article Snippet: For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used.

    Techniques: IA, Fluorescence, Stable Transfection, Expressing, Labeling

    Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP (BC-Alexa647). Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.

    Journal: Nature Communications

    Article Title: Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance

    doi: 10.1038/ncomms13334

    Figure Lengend Snippet: Histone H3 modifications stabilize centrochromatin for kinetochore maintenance. ( a ) Immunofluorescence images of 1C7 cells expressing CLIP-H3.3 and the indicated tetR-fusion proteins for 48 h. H3.3 was detected by staining for anti-CLIP (BC-Alexa647). Merged images represent the overlay of EYFP, mCherry/TMR-Star signals with Alexa647 (panel 4). ( b ) Quantification of fluorescence signals of HAC-associated CLIP-H3.3 staining in individual cells transfected with the indicated tetR-fusion constructs. Values for the HAC-associated CLIP-H3.3 signals were normalized for the mean of the H3.3 signals of the nuclei. ( c ) Representative images of 1C7 cells expressing Halo-CENP-A and the indicated tetR-fusion proteins for 48 h. Halo-CENP-A was detected with a Coumarin-tagged Halo ligand. Left panels show the loading of newly synthesized Halo-CENP-A and the right panels show levels of total Halo-CENP-A molecules at centromeres. ( d , e ) Quantification of Coumarin fluorescence signal associated with the HAC and normalized to the average signals at endogenous centromeres. Solid bars indicate the medians and error bars represent the s.e.m. N =three independent experiments. Scale bars, 10 μm.

    Article Snippet: CLIP-H3.3 was detected with benzylcytosine labelled with Alexa647 after fixation (NEB).

    Techniques: Immunofluorescence, Expressing, Cross-linking Immunoprecipitation, Staining, Fluorescence, HAC Assay, Transfection, Construct, Synthesized

    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

    Journal: Cell Chemical Biology

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

    doi: 10.1016/j.chembiol.2019.02.014

    Figure Lengend 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

    Article Snippet: SNAP-Tag® AlexaFluor 647 (AF647) and AlexaFluor 488 (AF488) were purchased from New England BioLabs (Massachusetts, USA).

    Techniques: 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

    Journal: Cell Chemical Biology

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

    doi: 10.1016/j.chembiol.2019.02.014

    Figure Lengend 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

    Article Snippet: SNAP-Tag® AlexaFluor 647 (AF647) and AlexaFluor 488 (AF488) were purchased from New England BioLabs (Massachusetts, USA).

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