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    Structured Review

    New England Biolabs clip cell block
    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of <t>CLIP-NRXN-1xnλ</t> and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of <t>SNAP-NLGN-1xnλ</t> and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.
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    Images

    1) Product Images from "Using high-throughput barcode sequencing to efficiently map connectomes"

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    Journal: bioRxiv

    doi: 10.1101/099093

    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.
    Figure Legend Snippet: Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Techniques Used: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.
    Figure Legend Snippet: PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.

    Techniques Used: Proximity Ligation Assay, Positive Control, Cross-linking Immunoprecipitation

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.
    Figure Legend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Techniques Used: Expressing, Staining, Cross-linking Immunoprecipitation

    (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.
    Figure Legend Snippet: (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.

    Techniques Used: Functional Assay, Cross-linking Immunoprecipitation, Immunoprecipitation

    2) Product Images from "Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell"

    Article Title: Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-10-0596

    CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p
    Figure Legend Snippet: CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Techniques Used: Immunofluorescence, Stable Transfection, Labeling

    3) Product Images from "In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi"

    Article Title: In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E21-03-0094

    Myo1b knockdown impaired early-stage trafficking of insulin granules from the TGN. 832/3 cells stably expressing proCpepSNAP were transfected with nontargeting siRNA (Scrambled) or Myo1b-targeting siRNA (siRNA1) and then pulse-labeled with SNAP-TMR for 20 min and chased for 2 h. The frequency distribution of TMR-labeled granules 0 to ≥6 µm (A) and 0 to 1 µm (B) from the TGN in control and Myo1b-kd cells at t = 2-h chase. Data represent the mean ± SEM of 27–31 cells per condition from n = 3 independent experiments. * P ≤ 0.05 by two-way ANOVA with Sidak posttest analysis. (C) Representative confocal images (three-dimensional projection from five z-stacks, DiAna) of 832/3 cells stably expressing proCpepSNAP treated with control or Myo1b-targeting siRNA and then pulse chased with SNAP-TMR (red) for 2 h before staining with anti-TGN38 antibody (cyan) and counterstaining with DAPI (blue). Scale bar, 10 µm.
    Figure Legend Snippet: Myo1b knockdown impaired early-stage trafficking of insulin granules from the TGN. 832/3 cells stably expressing proCpepSNAP were transfected with nontargeting siRNA (Scrambled) or Myo1b-targeting siRNA (siRNA1) and then pulse-labeled with SNAP-TMR for 20 min and chased for 2 h. The frequency distribution of TMR-labeled granules 0 to ≥6 µm (A) and 0 to 1 µm (B) from the TGN in control and Myo1b-kd cells at t = 2-h chase. Data represent the mean ± SEM of 27–31 cells per condition from n = 3 independent experiments. * P ≤ 0.05 by two-way ANOVA with Sidak posttest analysis. (C) Representative confocal images (three-dimensional projection from five z-stacks, DiAna) of 832/3 cells stably expressing proCpepSNAP treated with control or Myo1b-targeting siRNA and then pulse chased with SNAP-TMR (red) for 2 h before staining with anti-TGN38 antibody (cyan) and counterstaining with DAPI (blue). Scale bar, 10 µm.

    Techniques Used: Stable Transfection, Expressing, Transfection, Labeling, Staining

    Rescue of Myo1b siRNA–induced early-stage trafficking defects by reexpression of RNAi-resistant Myo1b in proCpepSNAP-expressing 832/3 cells. 832/3 cells stably expressing proCpepSNAP were transfected with control siRNA (Scrambled), Myo1b-targeting siRNA (siRNA1 ), or Myo1b-targeting siRNA plus RNAi-resistant pEGFP-Myo1b (siRNA1 + rescue) and then pulse-labeled with SNAP-TMR (red) for 20 min and chased for 2 h before confocal imaging. (A) Frequency distributions of TMR-labeled granule distances ≤2 and > 2 µm from the TGN are shown. Data represent the mean ± SEM of 26–31 cells per condition from n = 3 independent experiments. * P
    Figure Legend Snippet: Rescue of Myo1b siRNA–induced early-stage trafficking defects by reexpression of RNAi-resistant Myo1b in proCpepSNAP-expressing 832/3 cells. 832/3 cells stably expressing proCpepSNAP were transfected with control siRNA (Scrambled), Myo1b-targeting siRNA (siRNA1 ), or Myo1b-targeting siRNA plus RNAi-resistant pEGFP-Myo1b (siRNA1 + rescue) and then pulse-labeled with SNAP-TMR (red) for 20 min and chased for 2 h before confocal imaging. (A) Frequency distributions of TMR-labeled granule distances ≤2 and > 2 µm from the TGN are shown. Data represent the mean ± SEM of 26–31 cells per condition from n = 3 independent experiments. * P

    Techniques Used: Expressing, Stable Transfection, Transfection, Labeling, Imaging

    4) Product Images from "The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate"

    Article Title: The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    doi: 10.1080/14756366.2020.1841182

    Specificity of the Huisgen reaction. Gel-imaging analysis of SNAP- tag ® labelling by a chemo-enzymatic approach with BGSN3 and three different DBCO-derivative fluorophores. Protein (5 µM) was incubated with 5 µM of the azide-based BG for 60 min at 25 °C; then, an equimolar amount of DBCO-based substrate was added for the chemical click reaction, keeping the same time and temperature conditions. As control, SNAP- tag ® was incubated only with SVG (lane 1, signal marked with an asterisk).
    Figure Legend Snippet: Specificity of the Huisgen reaction. Gel-imaging analysis of SNAP- tag ® labelling by a chemo-enzymatic approach with BGSN3 and three different DBCO-derivative fluorophores. Protein (5 µM) was incubated with 5 µM of the azide-based BG for 60 min at 25 °C; then, an equimolar amount of DBCO-based substrate was added for the chemical click reaction, keeping the same time and temperature conditions. As control, SNAP- tag ® was incubated only with SVG (lane 1, signal marked with an asterisk).

    Techniques Used: Imaging, Incubation

    5) Product Images from "Using high-throughput barcode sequencing to efficiently map connectomes"

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    Journal: bioRxiv

    doi: 10.1101/099093

    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.
    Figure Legend Snippet: Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Techniques Used: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.
    Figure Legend Snippet: PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.

    Techniques Used: Proximity Ligation Assay, Positive Control, Cross-linking Immunoprecipitation

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.
    Figure Legend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Techniques Used: Expressing, Staining, Cross-linking Immunoprecipitation

    (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.
    Figure Legend Snippet: (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.

    Techniques Used: Functional Assay, Cross-linking Immunoprecipitation, Immunoprecipitation

    6) Product Images from "Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses"

    Article Title: Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2583-16.2017

    Densin accelerates the forward trafficking of Ca v 1.2 in transfected HEK293T cells. A , Cells expressing Ca v 1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B , Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C , Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.
    Figure Legend Snippet: Densin accelerates the forward trafficking of Ca v 1.2 in transfected HEK293T cells. A , Cells expressing Ca v 1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B , Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C , Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.

    Techniques Used: Transfection, Expressing, Labeling, Synthesized, Incubation, Fluorescence

    7) Product Images from "Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N"

    Article Title: Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N

    Journal: Genes & Development

    doi: 10.1101/gad.259432.115

    Specific loading of CENP-N in middle/late S phase (see also Supplemental Fig. S6). ( A ) Representative images of HeLa cells expressing SNAP-CENP-N in different cell cycle phases. SNAP-CENP-N was labeled with TMR-Star (red), and cell cycle phases were determined
    Figure Legend Snippet: Specific loading of CENP-N in middle/late S phase (see also Supplemental Fig. S6). ( A ) Representative images of HeLa cells expressing SNAP-CENP-N in different cell cycle phases. SNAP-CENP-N was labeled with TMR-Star (red), and cell cycle phases were determined

    Techniques Used: Expressing, Labeling

    8) Product Images from "H3.3 is deposited at centromeres in S phase as a placeholder for newly assembled CENP-A in G1 phase"

    Article Title: H3.3 is deposited at centromeres in S phase as a placeholder for newly assembled CENP-A in G1 phase

    Journal: Nucleus

    doi: 10.4161/nucl.2.2.15211

    Histone variants (H3.1 and H3.3) and histone modifications (H3K4me2) at centromeres in S phase. (A) Chromatin fibers showing replication of CENP-A and H3K4me2 domains at centromeres after a short (5 min) or long (5 h) pulse with EdU. Fibers are stained with anti-CENP-A antibody (green), anti-H3K4me2 antibody (red) and newly replicated DNA is marked with EdU (blue). Scale bar is 5 µM. Corresponding line plots illustrate the intensity of CENP-A (top) or H3K4me2 (bottom) with EdU along the centromere track after a short or long pulse with EdU. (B) Centromeric chromatin fibers from cells stably expressing H3.1-SNAP (top) and H3.3-SNAP (bottom). Total H3.1-SNAP or H3.3-SNAP is labeled with TMR (red), centromeres are marked with anti-CENP-A antibody (green), and newly replicated DNA is marked with EdU (blue, 1 hour pulse). Scale bar is 5 µM. (C) Quantitation shows that both H3.1-SNAP and H3.3-SNAP are mostly found in interspersed regions (red) between CENP-A and some overlap within CENP-A blocks (yellow), n=15 fibers for each cell line (mean ± SD).
    Figure Legend Snippet: Histone variants (H3.1 and H3.3) and histone modifications (H3K4me2) at centromeres in S phase. (A) Chromatin fibers showing replication of CENP-A and H3K4me2 domains at centromeres after a short (5 min) or long (5 h) pulse with EdU. Fibers are stained with anti-CENP-A antibody (green), anti-H3K4me2 antibody (red) and newly replicated DNA is marked with EdU (blue). Scale bar is 5 µM. Corresponding line plots illustrate the intensity of CENP-A (top) or H3K4me2 (bottom) with EdU along the centromere track after a short or long pulse with EdU. (B) Centromeric chromatin fibers from cells stably expressing H3.1-SNAP (top) and H3.3-SNAP (bottom). Total H3.1-SNAP or H3.3-SNAP is labeled with TMR (red), centromeres are marked with anti-CENP-A antibody (green), and newly replicated DNA is marked with EdU (blue, 1 hour pulse). Scale bar is 5 µM. (C) Quantitation shows that both H3.1-SNAP and H3.3-SNAP are mostly found in interspersed regions (red) between CENP-A and some overlap within CENP-A blocks (yellow), n=15 fibers for each cell line (mean ± SD).

    Techniques Used: Staining, Stable Transfection, Expressing, Labeling, Quantitation Assay

    Dilution and deposition of CENP-A visualized on chromatin fibers. (A) Chromatin fibers from S phase (EdU positive) or G 1 phase (EdU negative, synchronized in mid-G 1 by release from nocodazole block) cells stained with anti-CENP-A antibody. Cells were pulsed with EdU for 1 hour. DNA is stained with DAPI. Merge of CENP-A and DAPI is shown. Scale bar is 5 µM. (B) Quantitation of total CENP-A intensity measured using ImageJ (top) or total CENP-A spots (bottom) at centromeres in S phase (Cen S) compared to G 1 phase (Cen G 1 ), n=15 centromere fibers for each cell cycle stage (mean ± SD). (C) Newly synthesized CENP-A is deposited at centromeres in late telophase/early G 1 phase. Cells expressing CENP-A-SNAP were labeled with TMR only (for total CENP-A-SNAP, top), or quenched with BTP and then immediately labeled with TMR with no chase time (to assess blocking efficiency, middle), or quenched with BTP then allowed 6 hours chase time for new protein synthesis before labeling of new CENP-A-SNAP with TMR (bottom). Cells were co-stained with anti-tubulin antibody that marks the midbody (white arrow) as an indicator of late telophase/early G 1 phase cells. Scale bar is 15 µM. (D) (Upper part) Cell synchronization and experimental scheme for detection of newly synthesized CENP-A on chromatin fibers from G 1 phase cells. (Lower part) Chromatin fibers from G 1 cells were labeled with TMR to mark newly synthesized CENP-A-SNAP (red) and stained with anti-CENP-A antibody to mark total CENP-A (both total CENP-A-SNAP and endogenous CENP-A in green). 38 ± 13% of total CENP-A spots along the centromere track are new CENP-A-SNAP spots (n=20 fibers, mean ± SD). Scale bar is 5 µM.
    Figure Legend Snippet: Dilution and deposition of CENP-A visualized on chromatin fibers. (A) Chromatin fibers from S phase (EdU positive) or G 1 phase (EdU negative, synchronized in mid-G 1 by release from nocodazole block) cells stained with anti-CENP-A antibody. Cells were pulsed with EdU for 1 hour. DNA is stained with DAPI. Merge of CENP-A and DAPI is shown. Scale bar is 5 µM. (B) Quantitation of total CENP-A intensity measured using ImageJ (top) or total CENP-A spots (bottom) at centromeres in S phase (Cen S) compared to G 1 phase (Cen G 1 ), n=15 centromere fibers for each cell cycle stage (mean ± SD). (C) Newly synthesized CENP-A is deposited at centromeres in late telophase/early G 1 phase. Cells expressing CENP-A-SNAP were labeled with TMR only (for total CENP-A-SNAP, top), or quenched with BTP and then immediately labeled with TMR with no chase time (to assess blocking efficiency, middle), or quenched with BTP then allowed 6 hours chase time for new protein synthesis before labeling of new CENP-A-SNAP with TMR (bottom). Cells were co-stained with anti-tubulin antibody that marks the midbody (white arrow) as an indicator of late telophase/early G 1 phase cells. Scale bar is 15 µM. (D) (Upper part) Cell synchronization and experimental scheme for detection of newly synthesized CENP-A on chromatin fibers from G 1 phase cells. (Lower part) Chromatin fibers from G 1 cells were labeled with TMR to mark newly synthesized CENP-A-SNAP (red) and stained with anti-CENP-A antibody to mark total CENP-A (both total CENP-A-SNAP and endogenous CENP-A in green). 38 ± 13% of total CENP-A spots along the centromere track are new CENP-A-SNAP spots (n=20 fibers, mean ± SD). Scale bar is 5 µM.

    Techniques Used: Blocking Assay, Staining, Quantitation Assay, Synthesized, Expressing, Labeling

    New H3 deposition at centromeres in S phase. (A) Efficient ‘quenching’ of TMR (BTP, no chase, TMR) signal on whole cell nuclei in cells lines stably expressing H3.1-SNAP (left part) or H3.3-SNAP (right part). Anti-SNAP antibody (green) also recognizes SNAP that has been ‘quenched’ by BTP. Scale bar is 10 µM. (B) Experimental scheme to follow the incorporation of newly synthesized H3.1-SNAP or H3.3-SNAP on newly replicated DNA in S phase. (C) (Left part) On global non-centromeric chromatin, newly synthesized H3.1-SNAP is deposited on EdU positive fibers and is absent from EdU negative fibers. (Right part) On global chromatin, newly synthesized H3.3-SNAP is deposited on both EdU positive fibers and EdU negative fibers. Scale bar is 5 µM, n=20 fibers in each case. (D) Deposition of newly synthesized H3.1-SNAP at centromeres in S phase. Centromeres are marked with anti-CENP-A antibody (green), new H3.1-SNAP with TMR (red), newly replicated DNA with EdU (blue) and DNA is stained with DAPI. Merge of TMR, CENP-A and EdU is shown. Scale bar is 5 µM. Quantification (n = 16 fibers, mean ± SD) of the domain where new H3.1-SNAP is deposited reveals that new H3.1-SNAP is deposited at CENP-A domains (yellow, black arrows) or interspersed domains (red) with equal frequencies. (E) Deposition of newly synthesized H3.3-SNAP at centromeres in S phase. Centromeres are marked with anti-CENP-A antibody (green), new H3.3-SNAP with TMR (red), newly replicated DNA with EdU (blue) and DNA is stained with DAPI. Merge of TMR, CENP-A and EdU is shown. Scale bar is 5 µM. Quantification (n=16 fibers, mean ± SD) of the domain where new H3.3-SNAP is deposited reveals that new H3.3-SNAP is deposited more frequently in interspersed domains (red) than CENP-A domains (yellow, black arrows).
    Figure Legend Snippet: New H3 deposition at centromeres in S phase. (A) Efficient ‘quenching’ of TMR (BTP, no chase, TMR) signal on whole cell nuclei in cells lines stably expressing H3.1-SNAP (left part) or H3.3-SNAP (right part). Anti-SNAP antibody (green) also recognizes SNAP that has been ‘quenched’ by BTP. Scale bar is 10 µM. (B) Experimental scheme to follow the incorporation of newly synthesized H3.1-SNAP or H3.3-SNAP on newly replicated DNA in S phase. (C) (Left part) On global non-centromeric chromatin, newly synthesized H3.1-SNAP is deposited on EdU positive fibers and is absent from EdU negative fibers. (Right part) On global chromatin, newly synthesized H3.3-SNAP is deposited on both EdU positive fibers and EdU negative fibers. Scale bar is 5 µM, n=20 fibers in each case. (D) Deposition of newly synthesized H3.1-SNAP at centromeres in S phase. Centromeres are marked with anti-CENP-A antibody (green), new H3.1-SNAP with TMR (red), newly replicated DNA with EdU (blue) and DNA is stained with DAPI. Merge of TMR, CENP-A and EdU is shown. Scale bar is 5 µM. Quantification (n = 16 fibers, mean ± SD) of the domain where new H3.1-SNAP is deposited reveals that new H3.1-SNAP is deposited at CENP-A domains (yellow, black arrows) or interspersed domains (red) with equal frequencies. (E) Deposition of newly synthesized H3.3-SNAP at centromeres in S phase. Centromeres are marked with anti-CENP-A antibody (green), new H3.3-SNAP with TMR (red), newly replicated DNA with EdU (blue) and DNA is stained with DAPI. Merge of TMR, CENP-A and EdU is shown. Scale bar is 5 µM. Quantification (n=16 fibers, mean ± SD) of the domain where new H3.3-SNAP is deposited reveals that new H3.3-SNAP is deposited more frequently in interspersed domains (red) than CENP-A domains (yellow, black arrows).

    Techniques Used: Stable Transfection, Expressing, Synthesized, Staining

    Total H3.3 at centromeres is reduced in G 1 compared to S phase. (A) Experimental scheme for TMR labeling of total H3.1-SNAP or H3.3-SNAP at centromeres in S or G 1 phase cells. (B) Chromatin fibers showing total H3.3-SNAP (TMR) at centromeres (upper part), or global chromatin (lower part) in S phase (EdU positive), or G 1 phase (EdU negative). Centromeres are marked with anti-CENP-A antibody (green), total H3.3-SNAP is labeled with TMR (red), newly replicated DNA with EdU and DNA is stained with DAPI (gray). Scale bar is 5 µM. (C) Quantitation of total H3.3-SNAP (left part) or H3.1-SNAP (right part) intensity on chromatin fibers from centromeres or global chromatin in S and G 1 cell cycle phases. Total TMR intensity on centromere fibers marked by CENP-A staining (cen S or cen G 1 ) or on a non-centromeric fiber of equal length (global S or global G 1 ) was measured using ImageJ, n=16 fibers for each data set. The amount of H3.3-SNAP at centromeres drops significantly in G 1 (EdU negative) compared to S phase (EdU positive) (p=0.000038, Student's t test). (D) Quantitation of CENP-A spots (green), H3.3 spots (red) or CENP-A/H3.3 spots (yellow) at centromeres in S and G 1 phases, n=16 fibers, mean ± SD. The number of H3.3 spots and CENP-A/H3.3 spots at centromeres is reduced in G 1 phase compared to S phase.
    Figure Legend Snippet: Total H3.3 at centromeres is reduced in G 1 compared to S phase. (A) Experimental scheme for TMR labeling of total H3.1-SNAP or H3.3-SNAP at centromeres in S or G 1 phase cells. (B) Chromatin fibers showing total H3.3-SNAP (TMR) at centromeres (upper part), or global chromatin (lower part) in S phase (EdU positive), or G 1 phase (EdU negative). Centromeres are marked with anti-CENP-A antibody (green), total H3.3-SNAP is labeled with TMR (red), newly replicated DNA with EdU and DNA is stained with DAPI (gray). Scale bar is 5 µM. (C) Quantitation of total H3.3-SNAP (left part) or H3.1-SNAP (right part) intensity on chromatin fibers from centromeres or global chromatin in S and G 1 cell cycle phases. Total TMR intensity on centromere fibers marked by CENP-A staining (cen S or cen G 1 ) or on a non-centromeric fiber of equal length (global S or global G 1 ) was measured using ImageJ, n=16 fibers for each data set. The amount of H3.3-SNAP at centromeres drops significantly in G 1 (EdU negative) compared to S phase (EdU positive) (p=0.000038, Student's t test). (D) Quantitation of CENP-A spots (green), H3.3 spots (red) or CENP-A/H3.3 spots (yellow) at centromeres in S and G 1 phases, n=16 fibers, mean ± SD. The number of H3.3 spots and CENP-A/H3.3 spots at centromeres is reduced in G 1 phase compared to S phase.

    Techniques Used: Labeling, Staining, Quantitation Assay

    9) Product Images from "Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses"

    Article Title: Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Cav1.2 Ca2+ Channels at Excitatory Synapses

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2583-16.2017

    Densin accelerates the forward trafficking of Ca v 1.2 in transfected HEK293T cells. A , Cells expressing Ca v 1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B , Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C , Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.
    Figure Legend Snippet: Densin accelerates the forward trafficking of Ca v 1.2 in transfected HEK293T cells. A , Cells expressing Ca v 1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B , Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C , Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.

    Techniques Used: Transfection, Expressing, Labeling, Synthesized, Incubation, Fluorescence

    10) Product Images from "Distinct stages in the recognition, sorting and packaging of proTGFα into COPII coated transport vesicles"

    Article Title: Distinct stages in the recognition, sorting and packaging of proTGFα into COPII coated transport vesicles

    Journal: bioRxiv

    doi: 10.1101/040410

    Comparison of pro/HA-SNAP-TGFα localization and trafficking kinetics in WT and CNIH KO HeLa cells. (A) Immunofluorescence localization with anti-HA showing steady state localization of pro/HA-SNAP-TGFα in WT or CNIH KO cells as labeled. The steady state localization of pro/HA-SNAP-TGFα did not show a significant difference. (B) TMR-Star was used to label newly synthesized pro/HA-SNAP-TGFα in WT or CNIH KO cells as indicated. Cells were fixed for microscopy at the indicated time points. At 5min, the majority of newly synthesized pro/HA-SNAP-TGFα was localized in the ER, with some punctate distribution, possibly representing the ERGIC. At 15min and 30min the majority of TMR-Star signal was found in perinuclear punctate structures resembling the Golgi apparatus. At 60min, TMR-Star signal became visible at the plasma membrane, although significant signal was also observed in ER and Golgi-like punctate structures, likely representing newly synthesized pro/HA-SNAP-TGFα in earlier steps of maturation. The differences between pro/HA-SNAP-TGFα localization in WT and CNIH KO cells were subtle.
    Figure Legend Snippet: Comparison of pro/HA-SNAP-TGFα localization and trafficking kinetics in WT and CNIH KO HeLa cells. (A) Immunofluorescence localization with anti-HA showing steady state localization of pro/HA-SNAP-TGFα in WT or CNIH KO cells as labeled. The steady state localization of pro/HA-SNAP-TGFα did not show a significant difference. (B) TMR-Star was used to label newly synthesized pro/HA-SNAP-TGFα in WT or CNIH KO cells as indicated. Cells were fixed for microscopy at the indicated time points. At 5min, the majority of newly synthesized pro/HA-SNAP-TGFα was localized in the ER, with some punctate distribution, possibly representing the ERGIC. At 15min and 30min the majority of TMR-Star signal was found in perinuclear punctate structures resembling the Golgi apparatus. At 60min, TMR-Star signal became visible at the plasma membrane, although significant signal was also observed in ER and Golgi-like punctate structures, likely representing newly synthesized pro/HA-SNAP-TGFα in earlier steps of maturation. The differences between pro/HA-SNAP-TGFα localization in WT and CNIH KO cells were subtle.

    Techniques Used: Immunofluorescence, Labeling, Synthesized, Microscopy

    11) Product Images from "Cohesin can remain associated with chromosomes during DNA replication"

    Article Title: Cohesin can remain associated with chromosomes during DNA replication

    Journal: bioRxiv

    doi: 10.1101/124107

    Cohesin remains associated with the same area of chromatin over long time periods a) Live cell microscopy images of Scc1-Halo JF549 JF646 SNAP-H3.3 WAPL 1116-1119Δ U20S Cells before photobleaching with a 568nm laser line, immediately after photobleaching and 23 hours later. Scale bar, 5 μ m b) Live cell microscopy images of Scc1-Halo JF549 DY505 SNAP-H3.3 WAPL 1116-1119Δ U20S cells in mitosis. Scale bar, 2 μ m
    Figure Legend Snippet: Cohesin remains associated with the same area of chromatin over long time periods a) Live cell microscopy images of Scc1-Halo JF549 JF646 SNAP-H3.3 WAPL 1116-1119Δ U20S Cells before photobleaching with a 568nm laser line, immediately after photobleaching and 23 hours later. Scale bar, 5 μ m b) Live cell microscopy images of Scc1-Halo JF549 DY505 SNAP-H3.3 WAPL 1116-1119Δ U20S cells in mitosis. Scale bar, 2 μ m

    Techniques Used: Microscopy

    Pulse-Chase Frap (pcFRAP) permits observation of chromatin binding over long time periods. a) Immunoblot and In Gel Fluorescence of Scc1-Halo and SNAP-H3.3 U20S nuclear extract b) Live cell microscopy images of Scc1-Halo JF549 and DY505 SNAP-H3.3. Scale bar, 5 μ m c) Schematic shows how residual fluorescent HaloTag ligand labels newly synthesised HaloTag fusion proteins. Incubation with an unlabelled ligand permanently blocks new proteins from becoming labelled d) Average intensity projections of z-stacks from Scc1-Halo whole nuclear FRAP experiments. Scale bar, 5 μ m e) Mean fluorescence intensity of Scc1-Halo JF549 nuclei 16 hours after bleaching of whole nucleus relative to prebleach Intensity. Recovery was observed in the presence or absence of blocking HaloTag ligand. n=11 f ) Graph depicting half nuclear FRAP for JF549 SNAP-H3.3. n=6.
    Figure Legend Snippet: Pulse-Chase Frap (pcFRAP) permits observation of chromatin binding over long time periods. a) Immunoblot and In Gel Fluorescence of Scc1-Halo and SNAP-H3.3 U20S nuclear extract b) Live cell microscopy images of Scc1-Halo JF549 and DY505 SNAP-H3.3. Scale bar, 5 μ m c) Schematic shows how residual fluorescent HaloTag ligand labels newly synthesised HaloTag fusion proteins. Incubation with an unlabelled ligand permanently blocks new proteins from becoming labelled d) Average intensity projections of z-stacks from Scc1-Halo whole nuclear FRAP experiments. Scale bar, 5 μ m e) Mean fluorescence intensity of Scc1-Halo JF549 nuclei 16 hours after bleaching of whole nucleus relative to prebleach Intensity. Recovery was observed in the presence or absence of blocking HaloTag ligand. n=11 f ) Graph depicting half nuclear FRAP for JF549 SNAP-H3.3. n=6.

    Techniques Used: Pulse Chase, Binding Assay, Fluorescence, Microscopy, Incubation, Blocking Assay

    12) Product Images from "Reconstituting Drosophila Centromere Identity in Human Cells"

    Article Title: Reconstituting Drosophila Centromere Identity in Human Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2019.08.067

    Drosophila dCENP-A Can Be Propagated at Ectopic Sites on Human Chromosomes (A) Experimental procedure to test epigenetic inheritance of dCENP-A in human cells. 48 h (day 2) after transfection of Cal1-GFP-LacI and dCENP-A-SNAP, cells were either stained with TMR ∗ and subjected to IF or incubated with IPTG to remove CAL1-GFP-LacI binding and SNAP-Cell Block to quench existing SNAP-dCENP-A molecules. After further culturing for 24 h, cells were incubated with TMR-Star to stain newly synthesized SNAP-dCENP-A (day 3). (B and C) Representative IF images of the recruitment of dCENP-A to the LacO arrays 2 days (B) or 3 days (C) after transfection of CAL1-GFP-LacIw and dCENP-A-SNAP in U2OS-LacO stable cell line expressing all three Drosophila centromeric proteins or only dCENP-A. Insets show magnification of the boxed regions. Scale bar, 5 μm. (D) Quantification of the percentage of cells with TMR ∗ signal at the LacO using imaging data of (B) and (C). Two experiments (dark circles) and their average (column) are shown. ∗ p
    Figure Legend Snippet: Drosophila dCENP-A Can Be Propagated at Ectopic Sites on Human Chromosomes (A) Experimental procedure to test epigenetic inheritance of dCENP-A in human cells. 48 h (day 2) after transfection of Cal1-GFP-LacI and dCENP-A-SNAP, cells were either stained with TMR ∗ and subjected to IF or incubated with IPTG to remove CAL1-GFP-LacI binding and SNAP-Cell Block to quench existing SNAP-dCENP-A molecules. After further culturing for 24 h, cells were incubated with TMR-Star to stain newly synthesized SNAP-dCENP-A (day 3). (B and C) Representative IF images of the recruitment of dCENP-A to the LacO arrays 2 days (B) or 3 days (C) after transfection of CAL1-GFP-LacIw and dCENP-A-SNAP in U2OS-LacO stable cell line expressing all three Drosophila centromeric proteins or only dCENP-A. Insets show magnification of the boxed regions. Scale bar, 5 μm. (D) Quantification of the percentage of cells with TMR ∗ signal at the LacO using imaging data of (B) and (C). Two experiments (dark circles) and their average (column) are shown. ∗ p

    Techniques Used: Transfection, Staining, Incubation, Binding Assay, Blocking Assay, Synthesized, Stable Transfection, Expressing, Imaging

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    New England Biolabs clip cell block
    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of <t>CLIP-NRXN-1xnλ</t> and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of <t>SNAP-NLGN-1xnλ</t> and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.
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    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Article Snippet: Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.

    Techniques: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: PLA screen for interacting synPRE-P and synPOST-P proteins, part 1. (a) PLA can be used to probe interactions across neurons, for example detecting the interaction of the synaptic proteins MYC-NRXN1B and HA-NLGN1AB. These largely unmodified proteins result in a strong PLA signal across XONAs, acting as a positive control for our screen for synaptically targeted and interacting SYNseq proteins. (b) Addition of CLIP and SNAP domains to the extracellular domains of myc-NRXN1B and HA-NLGN1AB are well tolerated, as indicated by PLA signal across XONAs, when tested together with HA-NLGN1AB or myc-NRXN1B. Scale bar = 50 μm.

    Article Snippet: Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.

    Techniques: Proximity Ligation Assay, Positive Control, Cross-linking Immunoprecipitation

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Article Snippet: Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.

    Techniques: Expressing, Staining, Cross-linking Immunoprecipitation

    (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: (a) The BG-PEG-Biotin-PEG-BC crosslinker is equipped with functional groups BG and BC, which mediate the covalent tagging of SNAP or CLIP respectively. In addition, the molecule contains a biotin moiety for immunoprecipitation. (b) The BG-PEG-(S-S)-Biotin-BC crosslinker contains a cleavable disulfide bridge for non-denaturing elution in addition to the same functional groups as the BG-PEG-Biotin-BC crosslinker. We use the two crosslinkers interchangeably in this study, unless non-denaturing elution is required.

    Article Snippet: Briefly, we incubated the cells with 2.5 μM bifunctional crosslinker in complete neuron media for 30 min. We then blocked all unreacted SNAP and CLIP epitopes by incubating the cells in 10 μM each of SNAP-cell block and CLIP-cell block (NEB S9106S and S9220S) in full neuron media for 30 min. We then washed the cells three times with full neuron media and finally in PBS.

    Techniques: Functional Assay, Cross-linking Immunoprecipitation, Immunoprecipitation

    CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Journal: Molecular Biology of the Cell

    Article Title: Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

    doi: 10.1091/mbc.E17-10-0596

    Figure Lengend Snippet: CENP-C and CENP-N degradation has no significant effect on centromeric CENP-A maintenance. (A, B) Cell lines containing AID tagged CENP-N, CENP-C, or both were treated with IAA to degrade the indicated proteins. Blue bars represent cells not treated with IAA. Cells were maintained in IAA beginning just after mitosis (red bars) and harvested in an early S-phase thymidine (thym) arrest (A) or after mitosis (mustard bars) or in early S phase (green bars) and harvested in a G2-phase roscovitine (rosco) arrest (B). Centromeric CENP-A immunofluorescence signal was normalized to the no-IAA signal. (C) Degradation of CENP-N inhibits new CENP-A assembly but not preexisting CENP-A in chromatin. The CENP-N AID-sfGFP cell line containing a stably integrated SNAP-tagged CENP-A was either fluorescently labeled or quenched according to the schematic (left panel). Green bars represent the time of synthesis of the fluorescent population of SNAP-tagged CENP-A. IAA was added as in A. Centromeric TMR-Star intensity represents the fluorescent population of SNAP-tagged CENP-A. Data are presented as mean ± SEM for three independent replicates. * p

    Article Snippet: Cell labeling or blocking was done by incubating cells with 2 µM SNAP-Cell TMR-Star (New England BioLabs) or 6.7 µM SNAP-Cell Block (New England Biolabs) diluted with media for 15 min, washed with phosphate-buffered saline (PBS) + 1 mM MgCl2 and CaCl2 , incubated in media for 1 h, washed with PBS + 1 mM MgCl2 and CaCl2 , and put back in media until ready to harvest.

    Techniques: Immunofluorescence, Stable Transfection, Labeling

    Myo1b knockdown impaired early-stage trafficking of insulin granules from the TGN. 832/3 cells stably expressing proCpepSNAP were transfected with nontargeting siRNA (Scrambled) or Myo1b-targeting siRNA (siRNA1) and then pulse-labeled with SNAP-TMR for 20 min and chased for 2 h. The frequency distribution of TMR-labeled granules 0 to ≥6 µm (A) and 0 to 1 µm (B) from the TGN in control and Myo1b-kd cells at t = 2-h chase. Data represent the mean ± SEM of 27–31 cells per condition from n = 3 independent experiments. * P ≤ 0.05 by two-way ANOVA with Sidak posttest analysis. (C) Representative confocal images (three-dimensional projection from five z-stacks, DiAna) of 832/3 cells stably expressing proCpepSNAP treated with control or Myo1b-targeting siRNA and then pulse chased with SNAP-TMR (red) for 2 h before staining with anti-TGN38 antibody (cyan) and counterstaining with DAPI (blue). Scale bar, 10 µm.

    Journal: Molecular Biology of the Cell

    Article Title: In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

    doi: 10.1091/mbc.E21-03-0094

    Figure Lengend Snippet: Myo1b knockdown impaired early-stage trafficking of insulin granules from the TGN. 832/3 cells stably expressing proCpepSNAP were transfected with nontargeting siRNA (Scrambled) or Myo1b-targeting siRNA (siRNA1) and then pulse-labeled with SNAP-TMR for 20 min and chased for 2 h. The frequency distribution of TMR-labeled granules 0 to ≥6 µm (A) and 0 to 1 µm (B) from the TGN in control and Myo1b-kd cells at t = 2-h chase. Data represent the mean ± SEM of 27–31 cells per condition from n = 3 independent experiments. * P ≤ 0.05 by two-way ANOVA with Sidak posttest analysis. (C) Representative confocal images (three-dimensional projection from five z-stacks, DiAna) of 832/3 cells stably expressing proCpepSNAP treated with control or Myo1b-targeting siRNA and then pulse chased with SNAP-TMR (red) for 2 h before staining with anti-TGN38 antibody (cyan) and counterstaining with DAPI (blue). Scale bar, 10 µm.

    Article Snippet: SNAP-Cell TMR-Star (S9105S) and SNAP-Cell Block (S9106S) were obtained from New England Biolabs (Ipswich, MA).

    Techniques: Stable Transfection, Expressing, Transfection, Labeling, Staining

    Rescue of Myo1b siRNA–induced early-stage trafficking defects by reexpression of RNAi-resistant Myo1b in proCpepSNAP-expressing 832/3 cells. 832/3 cells stably expressing proCpepSNAP were transfected with control siRNA (Scrambled), Myo1b-targeting siRNA (siRNA1 ), or Myo1b-targeting siRNA plus RNAi-resistant pEGFP-Myo1b (siRNA1 + rescue) and then pulse-labeled with SNAP-TMR (red) for 20 min and chased for 2 h before confocal imaging. (A) Frequency distributions of TMR-labeled granule distances ≤2 and > 2 µm from the TGN are shown. Data represent the mean ± SEM of 26–31 cells per condition from n = 3 independent experiments. * P

    Journal: Molecular Biology of the Cell

    Article Title: In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

    doi: 10.1091/mbc.E21-03-0094

    Figure Lengend Snippet: Rescue of Myo1b siRNA–induced early-stage trafficking defects by reexpression of RNAi-resistant Myo1b in proCpepSNAP-expressing 832/3 cells. 832/3 cells stably expressing proCpepSNAP were transfected with control siRNA (Scrambled), Myo1b-targeting siRNA (siRNA1 ), or Myo1b-targeting siRNA plus RNAi-resistant pEGFP-Myo1b (siRNA1 + rescue) and then pulse-labeled with SNAP-TMR (red) for 20 min and chased for 2 h before confocal imaging. (A) Frequency distributions of TMR-labeled granule distances ≤2 and > 2 µm from the TGN are shown. Data represent the mean ± SEM of 26–31 cells per condition from n = 3 independent experiments. * P

    Article Snippet: SNAP-Cell TMR-Star (S9105S) and SNAP-Cell Block (S9106S) were obtained from New England Biolabs (Ipswich, MA).

    Techniques: Expressing, Stable Transfection, Transfection, Labeling, Imaging

    Specificity of the Huisgen reaction. Gel-imaging analysis of SNAP- tag ® labelling by a chemo-enzymatic approach with BGSN3 and three different DBCO-derivative fluorophores. Protein (5 µM) was incubated with 5 µM of the azide-based BG for 60 min at 25 °C; then, an equimolar amount of DBCO-based substrate was added for the chemical click reaction, keeping the same time and temperature conditions. As control, SNAP- tag ® was incubated only with SVG (lane 1, signal marked with an asterisk).

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    Article Title: The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate

    doi: 10.1080/14756366.2020.1841182

    Figure Lengend Snippet: Specificity of the Huisgen reaction. Gel-imaging analysis of SNAP- tag ® labelling by a chemo-enzymatic approach with BGSN3 and three different DBCO-derivative fluorophores. Protein (5 µM) was incubated with 5 µM of the azide-based BG for 60 min at 25 °C; then, an equimolar amount of DBCO-based substrate was added for the chemical click reaction, keeping the same time and temperature conditions. As control, SNAP- tag ® was incubated only with SVG (lane 1, signal marked with an asterisk).

    Article Snippet: SNAP-Vista® Green ( SVG ), SNAP Cell® Block ( SCB ), SNAP Cell® 430 ( SC430 ), BG-PEG-NH2 ( BGPA ), pSNAP-tag (m) plasmid, DNA restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs (USA).

    Techniques: Imaging, Incubation