p9310  (New England Biolabs)


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    Structured Review

    New England Biolabs p9310
    P9310, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    New England Biolabs rabbit anti snap tag
    Binding kinetics influence GLP-1R recycling. a Dissociation curve indicating FRET between FITC-agonist complexed with surface <t>SNAP-GLP-1R,</t> after inhibition of internalization, using NaN 3 and 2-deoxyglucose 9 (Supplementary Fig. 9 ), 30 min agonist exposure, washout, and exendin(9-39) blockade, n = 4. Unmodified (non-FITC) agonist b residence time (1/ k off ), c association rate constant ( k on ), and d affinity, measured by TR-FRET in competition with exendin-4-FITC, with internalization inhibitors as above, and calculated using competitive kinetic method 42 , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4. e Confocal fluorescence indicating co-localization of exendin-4-FITC or exendin-phe1-FITC with SNAP-GLP-1R (labeled with SNAP-Surface-549) after 60 min agonist exposure in MIN6B1-SNAP-GLP-1R cells, representative images from n = 2 experiments; scale bars, 8 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Schematic illustrating endosomal binding protocol. SA-Tb streptavidin-terbium cryptate. g Real-time FRET measurement of FITC-agonist complexed with internalized SNAP-GLP-1R after 30 min agonist exposure, washout, exendin(9-39) blockade, and cleavage of SNAP-biotin from surface SNAP-GLP-1R with MesNa, n = 5. Exendin-4 h residence time and i association rate constant ± 10 μM BETP, measured by TR-FRET in competition with exendin-4-FITC, n = 4, paired t -test. Exendin-4-induced j internalization (30 min), and k recycling (60 min) ± 3 μM BETP, n = 4, paired t -test. l Prolonged insulin secretion with exendin-4 ± 3 µM BETP in INS-1 832/3 cells, 16 h, n = 5, paired t -test for E max assessed by four-parameter fit. Exendin-4 m cAMP, and n <t>β-arrestin-2</t> responses, in PathHunter CHO-GLP-1R cells ± 3 μM BETP, n = 3. Agonists applied at 100 nM, except where indicated, and performed in CHO-SNAP-GLP-1R cells, except where indicated. * p
    Rabbit Anti Snap Tag, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    94
    New England Biolabs anti snap tag antibody polyclonal
    Identifying the minimal binding region of LHBS and PKM2. (A) Total cell lysates of HuH-7 cells transfected with pHBV3.6 plasmid were harvested for affinity immunoprecipitations using control immunoglobulin (IgG), monoclonal (mAb, clone 86H6) and <t>polyclonal</t> (pAb, Abnova) anti-HBS antibodies. The resulted immunoprecipitants were probed with anti-HBS mAb (86H6) and anti-PKM2 antibodies. Representative immunoblots were shown. (B) 293T cells were transiently transfected with expression constructs of HA-PKM2 and <t>SNAP-tagged-LHBS,</t> -MHBS, -SHBS, and -PreS, as indicated, for 2 days, and total cell lysates were harvested for affinity immunoprecipitation using a mouse anti-HA antibody. Representative immunoblots using anti-PKM2 and anti-SNAP antibodies were shown. (C) Immortalized hepatocytes stably expressing LHBS were transfected with expression constructs of HA-PKM2 encoding different truncation fragments as indicated. Total cell lysates were harvested at 2 days post transfection and subjected to affinity immunoprecipitation using anti-HA antibody. Representative immunoblots of HA-PKM2 and LHBS detected by anti-HA and anti-LHBS/PreS1 antibodies were shown. *denotes the high exposure image of the low molecular weight protein HA-367-476.
    Anti Snap Tag Antibody Polyclonal, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti snap tag antibody polyclonal/product/New England Biolabs
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    Binding kinetics influence GLP-1R recycling. a Dissociation curve indicating FRET between FITC-agonist complexed with surface SNAP-GLP-1R, after inhibition of internalization, using NaN 3 and 2-deoxyglucose 9 (Supplementary Fig. 9 ), 30 min agonist exposure, washout, and exendin(9-39) blockade, n = 4. Unmodified (non-FITC) agonist b residence time (1/ k off ), c association rate constant ( k on ), and d affinity, measured by TR-FRET in competition with exendin-4-FITC, with internalization inhibitors as above, and calculated using competitive kinetic method 42 , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4. e Confocal fluorescence indicating co-localization of exendin-4-FITC or exendin-phe1-FITC with SNAP-GLP-1R (labeled with SNAP-Surface-549) after 60 min agonist exposure in MIN6B1-SNAP-GLP-1R cells, representative images from n = 2 experiments; scale bars, 8 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Schematic illustrating endosomal binding protocol. SA-Tb streptavidin-terbium cryptate. g Real-time FRET measurement of FITC-agonist complexed with internalized SNAP-GLP-1R after 30 min agonist exposure, washout, exendin(9-39) blockade, and cleavage of SNAP-biotin from surface SNAP-GLP-1R with MesNa, n = 5. Exendin-4 h residence time and i association rate constant ± 10 μM BETP, measured by TR-FRET in competition with exendin-4-FITC, n = 4, paired t -test. Exendin-4-induced j internalization (30 min), and k recycling (60 min) ± 3 μM BETP, n = 4, paired t -test. l Prolonged insulin secretion with exendin-4 ± 3 µM BETP in INS-1 832/3 cells, 16 h, n = 5, paired t -test for E max assessed by four-parameter fit. Exendin-4 m cAMP, and n β-arrestin-2 responses, in PathHunter CHO-GLP-1R cells ± 3 μM BETP, n = 3. Agonists applied at 100 nM, except where indicated, and performed in CHO-SNAP-GLP-1R cells, except where indicated. * p

    Journal: Nature Communications

    Article Title: Targeting GLP-1 receptor trafficking to improve agonist efficacy

    doi: 10.1038/s41467-018-03941-2

    Figure Lengend Snippet: Binding kinetics influence GLP-1R recycling. a Dissociation curve indicating FRET between FITC-agonist complexed with surface SNAP-GLP-1R, after inhibition of internalization, using NaN 3 and 2-deoxyglucose 9 (Supplementary Fig. 9 ), 30 min agonist exposure, washout, and exendin(9-39) blockade, n = 4. Unmodified (non-FITC) agonist b residence time (1/ k off ), c association rate constant ( k on ), and d affinity, measured by TR-FRET in competition with exendin-4-FITC, with internalization inhibitors as above, and calculated using competitive kinetic method 42 , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4. e Confocal fluorescence indicating co-localization of exendin-4-FITC or exendin-phe1-FITC with SNAP-GLP-1R (labeled with SNAP-Surface-549) after 60 min agonist exposure in MIN6B1-SNAP-GLP-1R cells, representative images from n = 2 experiments; scale bars, 8 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Schematic illustrating endosomal binding protocol. SA-Tb streptavidin-terbium cryptate. g Real-time FRET measurement of FITC-agonist complexed with internalized SNAP-GLP-1R after 30 min agonist exposure, washout, exendin(9-39) blockade, and cleavage of SNAP-biotin from surface SNAP-GLP-1R with MesNa, n = 5. Exendin-4 h residence time and i association rate constant ± 10 μM BETP, measured by TR-FRET in competition with exendin-4-FITC, n = 4, paired t -test. Exendin-4-induced j internalization (30 min), and k recycling (60 min) ± 3 μM BETP, n = 4, paired t -test. l Prolonged insulin secretion with exendin-4 ± 3 µM BETP in INS-1 832/3 cells, 16 h, n = 5, paired t -test for E max assessed by four-parameter fit. Exendin-4 m cAMP, and n β-arrestin-2 responses, in PathHunter CHO-GLP-1R cells ± 3 μM BETP, n = 3. Agonists applied at 100 nM, except where indicated, and performed in CHO-SNAP-GLP-1R cells, except where indicated. * p

    Article Snippet: Antibodies were primaries rabbit anti-β-arrestin-1/2 (D24H9, Cell Signaling, 1/1000), rabbit anti-SNAP tag (New England Biolabs, 1/500), mouse anti-α-tubulin (T5168, Sigma, 1/1000), rabbit anti-β-actin (4970, Cell Signaling, 1/1000), and mouse anti-GAPDH (6C5, Merck, 1/10,000); and IgG-HRP secondaries (Santa Cruz Biotechnology).

    Techniques: Binding Assay, Inhibition, Blocking Assay, Fluorescence, Labeling

    Comparison with licensed GLP-1R agonists. a Agonist residence time, measured by TR-FRET in competition with exendin-4-FITC in CHO-SNAP-GLP-1R cells, normalized to exendin-4-FITC response per assay, n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4 (ex-phe1 not included in statistical analysis). Agonist-induced b cAMP, and c β-arrestin-2 responses in PathHunter CHO-GLP-1R cells, 90 min, n = 5. d Bias calculated from data in b , c , 95% CI shown. Agonist-induced e internalization (60 min), and f recycling (60 min, measured in presence of 10 μM exendin(9-39) to block rebinding) in CHO-SNAP-GLP-1R cells, normalized to GLP-1 response per assay , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4 (ex-phe1 not included in statistical analysis). Note that exendin-phe1 results, from a different set of experiments, are shown in a and d – f , for purposes of comparison, after normalization to a reference ligand on a per assay basis. g Principal component analysis taking into account agonist k off , Δlog ( τ / K A ) for cAMP and β-arrestin-2, internalization (60 min), and recycling (60 min). h Prolonged insulin secretion in INS-1 832/3 cells, 16 h, n = 6, one-way randomized block ANOVA with Dunnett’s test vs. exendin-phe1. Agonists applied at 100 nM, except where indicated. * p

    Journal: Nature Communications

    Article Title: Targeting GLP-1 receptor trafficking to improve agonist efficacy

    doi: 10.1038/s41467-018-03941-2

    Figure Lengend Snippet: Comparison with licensed GLP-1R agonists. a Agonist residence time, measured by TR-FRET in competition with exendin-4-FITC in CHO-SNAP-GLP-1R cells, normalized to exendin-4-FITC response per assay, n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4 (ex-phe1 not included in statistical analysis). Agonist-induced b cAMP, and c β-arrestin-2 responses in PathHunter CHO-GLP-1R cells, 90 min, n = 5. d Bias calculated from data in b , c , 95% CI shown. Agonist-induced e internalization (60 min), and f recycling (60 min, measured in presence of 10 μM exendin(9-39) to block rebinding) in CHO-SNAP-GLP-1R cells, normalized to GLP-1 response per assay , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4 (ex-phe1 not included in statistical analysis). Note that exendin-phe1 results, from a different set of experiments, are shown in a and d – f , for purposes of comparison, after normalization to a reference ligand on a per assay basis. g Principal component analysis taking into account agonist k off , Δlog ( τ / K A ) for cAMP and β-arrestin-2, internalization (60 min), and recycling (60 min). h Prolonged insulin secretion in INS-1 832/3 cells, 16 h, n = 6, one-way randomized block ANOVA with Dunnett’s test vs. exendin-phe1. Agonists applied at 100 nM, except where indicated. * p

    Article Snippet: Antibodies were primaries rabbit anti-β-arrestin-1/2 (D24H9, Cell Signaling, 1/1000), rabbit anti-SNAP tag (New England Biolabs, 1/500), mouse anti-α-tubulin (T5168, Sigma, 1/1000), rabbit anti-β-actin (4970, Cell Signaling, 1/1000), and mouse anti-GAPDH (6C5, Merck, 1/10,000); and IgG-HRP secondaries (Santa Cruz Biotechnology).

    Techniques: Blocking Assay

    β-arrestin-biased signaling reduces insulin secretion. a Agonist-induced cAMP, β-arrestin-1 (βarr1), and β-arrestin-2 (βarr2) responses in PathHunter CHO-GLP-1R cells, 10 min incubation, n = 4–6; the four-parameter logistic fit of averaged data shown. b Web of bias, depicting relative pathway preference for each agonist; data represent the inverse logarithm of normalized log ( τ / K A ) values derived from a normalized to a reference agonist (exendin-4) and a reference pathway (cAMP); for further details, see Methods. Note that, β-arrestin-1 log ( τ / K A ) values for exendin-phe1 could not be calculated due to absence of detectable response. c , d as for a , b but for 90 min incubation. e Confocal images of MIN6B1-SNAP-GLP-1R cells transiently expressing β-arrestin-2-GFP, labeled with SNAP-Surface-549, and treated with indicated agonist for 5 min before fixation, representative images from n = 2 experiments; scale bars, 10 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Relationship between biased signaling (Supplementary Fig. 6 ) in PathHunter CHO-GLP-1R cells and maximal prolonged insulin secretion (Supplementary Fig. 1 ) in INS-1 832/3 cells. Association quantified by linear regression. g Effect of dual β-arrestin silencing on prolonged (16 h) exendin-4-induced insulin secretion in INS-1 832/3 cells, n = 4, paired t -test comparing E max . h As for g , but in MIN6B1 cells, n = 5, paired t -test. i As for g , but in EndoC-βH1 cells with stable knockdown of β-arrestin-1 and -2 by lentiviral transduction of shRNAs, n = 5, paired t -test. Agonists applied at 100 nM, except where indicated. * p

    Journal: Nature Communications

    Article Title: Targeting GLP-1 receptor trafficking to improve agonist efficacy

    doi: 10.1038/s41467-018-03941-2

    Figure Lengend Snippet: β-arrestin-biased signaling reduces insulin secretion. a Agonist-induced cAMP, β-arrestin-1 (βarr1), and β-arrestin-2 (βarr2) responses in PathHunter CHO-GLP-1R cells, 10 min incubation, n = 4–6; the four-parameter logistic fit of averaged data shown. b Web of bias, depicting relative pathway preference for each agonist; data represent the inverse logarithm of normalized log ( τ / K A ) values derived from a normalized to a reference agonist (exendin-4) and a reference pathway (cAMP); for further details, see Methods. Note that, β-arrestin-1 log ( τ / K A ) values for exendin-phe1 could not be calculated due to absence of detectable response. c , d as for a , b but for 90 min incubation. e Confocal images of MIN6B1-SNAP-GLP-1R cells transiently expressing β-arrestin-2-GFP, labeled with SNAP-Surface-549, and treated with indicated agonist for 5 min before fixation, representative images from n = 2 experiments; scale bars, 10 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Relationship between biased signaling (Supplementary Fig. 6 ) in PathHunter CHO-GLP-1R cells and maximal prolonged insulin secretion (Supplementary Fig. 1 ) in INS-1 832/3 cells. Association quantified by linear regression. g Effect of dual β-arrestin silencing on prolonged (16 h) exendin-4-induced insulin secretion in INS-1 832/3 cells, n = 4, paired t -test comparing E max . h As for g , but in MIN6B1 cells, n = 5, paired t -test. i As for g , but in EndoC-βH1 cells with stable knockdown of β-arrestin-1 and -2 by lentiviral transduction of shRNAs, n = 5, paired t -test. Agonists applied at 100 nM, except where indicated. * p

    Article Snippet: Antibodies were primaries rabbit anti-β-arrestin-1/2 (D24H9, Cell Signaling, 1/1000), rabbit anti-SNAP tag (New England Biolabs, 1/500), mouse anti-α-tubulin (T5168, Sigma, 1/1000), rabbit anti-β-actin (4970, Cell Signaling, 1/1000), and mouse anti-GAPDH (6C5, Merck, 1/10,000); and IgG-HRP secondaries (Santa Cruz Biotechnology).

    Techniques: Incubation, Derivative Assay, Expressing, Labeling, Transduction

    Identifying the minimal binding region of LHBS and PKM2. (A) Total cell lysates of HuH-7 cells transfected with pHBV3.6 plasmid were harvested for affinity immunoprecipitations using control immunoglobulin (IgG), monoclonal (mAb, clone 86H6) and polyclonal (pAb, Abnova) anti-HBS antibodies. The resulted immunoprecipitants were probed with anti-HBS mAb (86H6) and anti-PKM2 antibodies. Representative immunoblots were shown. (B) 293T cells were transiently transfected with expression constructs of HA-PKM2 and SNAP-tagged-LHBS, -MHBS, -SHBS, and -PreS, as indicated, for 2 days, and total cell lysates were harvested for affinity immunoprecipitation using a mouse anti-HA antibody. Representative immunoblots using anti-PKM2 and anti-SNAP antibodies were shown. (C) Immortalized hepatocytes stably expressing LHBS were transfected with expression constructs of HA-PKM2 encoding different truncation fragments as indicated. Total cell lysates were harvested at 2 days post transfection and subjected to affinity immunoprecipitation using anti-HA antibody. Representative immunoblots of HA-PKM2 and LHBS detected by anti-HA and anti-LHBS/PreS1 antibodies were shown. *denotes the high exposure image of the low molecular weight protein HA-367-476.

    Journal: PLoS Pathogens

    Article Title: Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2

    doi: 10.1371/journal.ppat.1008866

    Figure Lengend Snippet: Identifying the minimal binding region of LHBS and PKM2. (A) Total cell lysates of HuH-7 cells transfected with pHBV3.6 plasmid were harvested for affinity immunoprecipitations using control immunoglobulin (IgG), monoclonal (mAb, clone 86H6) and polyclonal (pAb, Abnova) anti-HBS antibodies. The resulted immunoprecipitants were probed with anti-HBS mAb (86H6) and anti-PKM2 antibodies. Representative immunoblots were shown. (B) 293T cells were transiently transfected with expression constructs of HA-PKM2 and SNAP-tagged-LHBS, -MHBS, -SHBS, and -PreS, as indicated, for 2 days, and total cell lysates were harvested for affinity immunoprecipitation using a mouse anti-HA antibody. Representative immunoblots using anti-PKM2 and anti-SNAP antibodies were shown. (C) Immortalized hepatocytes stably expressing LHBS were transfected with expression constructs of HA-PKM2 encoding different truncation fragments as indicated. Total cell lysates were harvested at 2 days post transfection and subjected to affinity immunoprecipitation using anti-HA antibody. Representative immunoblots of HA-PKM2 and LHBS detected by anti-HA and anti-LHBS/PreS1 antibodies were shown. *denotes the high exposure image of the low molecular weight protein HA-367-476.

    Article Snippet: AntibodiesThe following primary antibodies were used in this study: rabbit anti-PKM2 (CST 4053, Cell Signaling Technology), rabbit anti-HBsAg (ad/ay, PAB13969, Abnova), normal mouse IgG (sc-2025, Santa Cruz Biotechnology), normal rabbit IgG (sc-2027, Santa Cruz Biotechnology), rabbit anti-HBcAg (B0586, DAKO), mouse anti-HA-tag (sc-7392, Santa Cruz Biotechnology), mouse anti-HSC70 (sc-7298, Santa Cruz Biotechnology), rabbit anti-SNAP-tag (P9310, New England BioLabs), rabbit anti-beta-tubulin (NB600-936, Novus Biologicals), mouse anti-beta-actin (NB600-501, Novus Biologicals), rabbit anti-GAPDH (GTX100118, GeneTex), rabbit anti-Grp78 (GTX113340, GeneTex), rabbit anti-PLK1 (GTX104302, GeneTex), rabbit anti-MAD2L1 (GTX104680, GeneTex), rabbit anti-Bcl-2 (GTX100064, GeneTex), and rabbit anti-PCNA (GTX100539, GeneTex).

    Techniques: Binding Assay, Transfection, Plasmid Preparation, Western Blot, Expressing, Construct, Immunoprecipitation, Stable Transfection, Molecular Weight

    Establishment of a System for Real-Time Imaging of Pol δ Holoenzyme Assembly and Disassembly in Living Human Cells (A) Pol δ holoenzyme complexes formed by Pol δ3 and Pol δ4 assemblies and PCNA trimer ring. (B) Schematic diagram of SNAP-Pol δ p125 and Halo-PCNA fusion proteins (PIP, PCNA interacting protein; IDCL, Interdomain connecting loop). (C) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible Halo-PCNA. The percentage of the total PCNA protein represented by Halo-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (D) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible SNAP-Pol δ p125. The percentage of the total Pol δ p125 protein represented by SNAP-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (E) Growth curves of LOX stable cell lines co-expressing SNAP-Pol δ p125 and Halo-PCNA proteins. Cultures of two clonal lines, C3 (91% of total Pol δ p125 is SNAP-tagged) and C11 (50% of total Pol δ p125 SNAP-tagged), were grown in the absence or presence (+ dox) of 1 μg/ml doxycycline, and cells counted at time points indicated (cell number is relative to initial seeding). Values were obtained from two independent cultures. Error bars indicate SD.

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: Establishment of a System for Real-Time Imaging of Pol δ Holoenzyme Assembly and Disassembly in Living Human Cells (A) Pol δ holoenzyme complexes formed by Pol δ3 and Pol δ4 assemblies and PCNA trimer ring. (B) Schematic diagram of SNAP-Pol δ p125 and Halo-PCNA fusion proteins (PIP, PCNA interacting protein; IDCL, Interdomain connecting loop). (C) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible Halo-PCNA. The percentage of the total PCNA protein represented by Halo-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (D) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible SNAP-Pol δ p125. The percentage of the total Pol δ p125 protein represented by SNAP-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (E) Growth curves of LOX stable cell lines co-expressing SNAP-Pol δ p125 and Halo-PCNA proteins. Cultures of two clonal lines, C3 (91% of total Pol δ p125 is SNAP-tagged) and C11 (50% of total Pol δ p125 SNAP-tagged), were grown in the absence or presence (+ dox) of 1 μg/ml doxycycline, and cells counted at time points indicated (cell number is relative to initial seeding). Values were obtained from two independent cultures. Error bars indicate SD.

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Imaging, Stable Transfection, Expressing

    Pol δ’s Enzymatic Activity Regulates the Assembly and Disassembly Dynamics of the Holoenzyme (A) Schematic diagram of SNAP-Pol δ Dead p125 fusion protein (PIP, PCNA interacting protein). In this mutant, two essential active site residues that catalyze DNA synthesis (Asp602 and Asp757) and a residue essential for the 3′ –5′ exonuclease proofreading activity of Pol δ (Asp402) were mutated to alanine, resulting in a catalytically dead protein. (B) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible SNAP-Pol δ Dead p125. The percentage of the total Pol δ p125 protein represented by SNAP-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (C) Growth curve of LOX stable cell lines co-expressing SNAP-Pol δ Dead p125 and Halo-PCNA proteins. Cultures of three clonal lines, C2, C5, and C7 (82%, 52%m and 20% of total Pol δ p125 is SNAP-tagged, respectively), were grown in the absence or presence (+ dox) of 1 μg/ml doxycycline and cells counted at time points indicated (cell number is relative to initial seeding). Values were obtained from two independent cultures. Error bars indicate SD. (D) Histogram of the arrival and departure order of SNAP-Pol δ and Halo-PCNA molecules during 188 observed nuclear colocalization events (from SNAP-Pol δ Dead line C2). The percentage of SNAP-Pol δ and Halo-PCNA molecules arriving and departing first or at the same time are indicated. Error bars indicate SD as determined by Bootstrap analysis (1,000 iterations) of observed colocalizations. A 2-fold greater percentage of SNAP-Pol δ Dead molecules arrive before Halo-PCNA and depart after PCNA than SNAP-Pol δ WT. (E) Scatterplots of SNAP-Pol δ arrival (binding) and departure (dissociation) relative to Halo-PCNA binding and dissociation during nuclear colocalization events. Timelines (blue and orange-brown bars) shown in each quadrant indicate the binding order of SNAP-Pol δ and Halo-PCNA and time interval between arrival and departure of Pol δ/PCNA, as described in Figure 3 . The 95% confidence interval of median times is reported in Figure S2 . (F) Boxplots comparing the duration of Halo-PCNA colocalizations with SNAP-Pol δ WT (C11) versus SNAP-Pol δ Dead (C2). A total of 59 nuclei from SNAP-Pol δ WT cells from 4 independent experiments and 13 nuclei from SNAP-Pol δ Dead cells from 2 independent experiments were analyzed for colocalization. A Kruskal-Wallis test was used to determine pairwise significance. ****p = 2.21 × 10 −6 .

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: Pol δ’s Enzymatic Activity Regulates the Assembly and Disassembly Dynamics of the Holoenzyme (A) Schematic diagram of SNAP-Pol δ Dead p125 fusion protein (PIP, PCNA interacting protein). In this mutant, two essential active site residues that catalyze DNA synthesis (Asp602 and Asp757) and a residue essential for the 3′ –5′ exonuclease proofreading activity of Pol δ (Asp402) were mutated to alanine, resulting in a catalytically dead protein. (B) Immunoblot of cell lysates of LOX cell lines stably expressing doxycycline-inducible SNAP-Pol δ Dead p125. The percentage of the total Pol δ p125 protein represented by SNAP-tagged (top band) and untagged endogenous (bottom band) proteins is shown below each lane. (C) Growth curve of LOX stable cell lines co-expressing SNAP-Pol δ Dead p125 and Halo-PCNA proteins. Cultures of three clonal lines, C2, C5, and C7 (82%, 52%m and 20% of total Pol δ p125 is SNAP-tagged, respectively), were grown in the absence or presence (+ dox) of 1 μg/ml doxycycline and cells counted at time points indicated (cell number is relative to initial seeding). Values were obtained from two independent cultures. Error bars indicate SD. (D) Histogram of the arrival and departure order of SNAP-Pol δ and Halo-PCNA molecules during 188 observed nuclear colocalization events (from SNAP-Pol δ Dead line C2). The percentage of SNAP-Pol δ and Halo-PCNA molecules arriving and departing first or at the same time are indicated. Error bars indicate SD as determined by Bootstrap analysis (1,000 iterations) of observed colocalizations. A 2-fold greater percentage of SNAP-Pol δ Dead molecules arrive before Halo-PCNA and depart after PCNA than SNAP-Pol δ WT. (E) Scatterplots of SNAP-Pol δ arrival (binding) and departure (dissociation) relative to Halo-PCNA binding and dissociation during nuclear colocalization events. Timelines (blue and orange-brown bars) shown in each quadrant indicate the binding order of SNAP-Pol δ and Halo-PCNA and time interval between arrival and departure of Pol δ/PCNA, as described in Figure 3 . The 95% confidence interval of median times is reported in Figure S2 . (F) Boxplots comparing the duration of Halo-PCNA colocalizations with SNAP-Pol δ WT (C11) versus SNAP-Pol δ Dead (C2). A total of 59 nuclei from SNAP-Pol δ WT cells from 4 independent experiments and 13 nuclei from SNAP-Pol δ Dead cells from 2 independent experiments were analyzed for colocalization. A Kruskal-Wallis test was used to determine pairwise significance. ****p = 2.21 × 10 −6 .

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Activity Assay, Mutagenesis, DNA Synthesis, Stable Transfection, Expressing, Binding Assay

    The Catalytic Activity of Pol δ Affects Its Residence Time on Chromatin (A) Residence times were determined from 1–cumulative distribution function (1–CDF) plots of nuclear SNAP-Pol δ WT and Dead bound to chromatin (representative examples shown) fitted to a single (gray dashed) or two-component (red solid) exponential decay model. These curves identified two predominant populations: one of short (

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: The Catalytic Activity of Pol δ Affects Its Residence Time on Chromatin (A) Residence times were determined from 1–cumulative distribution function (1–CDF) plots of nuclear SNAP-Pol δ WT and Dead bound to chromatin (representative examples shown) fitted to a single (gray dashed) or two-component (red solid) exponential decay model. These curves identified two predominant populations: one of short (

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Activity Assay

    Pol δ Holoenzyme Assembly and Disassembly on the Genome Follow a Predominant Order (A) Potential order of SNAP-Pol δ and Halo-PCNA arrival and departure at sites of genome binding. Top: three sequence combinations in which Pol δ and PCNA can arrive on the primed DNA strand, namely, PCNA arrives first, Pol δ arrives first, or they both arrive simultaneously. Bottom: departure of Pol δ or PCNA could occur in one of the three ways, namely, Pol δ could depart first, PCNA could depart first, or the two could leave simultaneously. (B) An example of an image series of a typical colocalization binding event observed by dual-color SMT. In this particular event, colocalization occurred from frames 3–7 (~6-s duration). Note the Halo-PCNA signal appears before the SNAP-Pol δ signal and disappears after the Pol δ signal. Scale bars represent 500 nm. (C) Histogram of the arrival and departure order of SNAP-Pol δ and Halo-PCNA molecules during observed genomic colocalization events. The percentage of SNAP-Pol δ and Halo-PCNA molecules arriving and departing first or at the same time are indicated. Error bars indicate SD, as determined by Bootstrap analysis (1,000 iterations) of observed colocalizations. The predominant order of events observed is that PCNA loading precedes Pol δ binding, followed by Pol δ dissociation preceding PCNA unloading. (D) Scatterplot of SNAP-Pol δ arrival (binding) and departure (dissociation) relative to Halo-PCNA binding and dissociation during nuclear colocalization events. Timelines (blue and orange bars) shown in each quadrant indicate the binding order of SNAP-Pol δ and Halo-PCNA and time interval between arrival and departure of Pol δ/PCNA for colocalizations within that quadrant (SNAP-Pol δ arriving/departing before/ after Halo-PCNA). Blue bars indicate the presence of genome-bound Halo-PCNA during colocalization events; orange bars indicate the presence of genome-bound SNAP-Pol δ (p125) during colocalization events. Median time intervals (s) between arrival (before colocalization) and departure (after colocalization) of PCNA/Pol δ are shown below bars; median time interval (s) of colocalization shown above bars. The 95% confidence interval of median times is reported in Figure S2 . Percentage of total colocalizations represented by the specific order of assembly/disassembly in each quadrant is indicated under timelines. Plots reveal multiple assembly/disassembly pathways of the holoenzyme. A total of 270 colocalization events from 59 nuclei of LOX C11 cells from 4 independent experiments were analyzed.

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: Pol δ Holoenzyme Assembly and Disassembly on the Genome Follow a Predominant Order (A) Potential order of SNAP-Pol δ and Halo-PCNA arrival and departure at sites of genome binding. Top: three sequence combinations in which Pol δ and PCNA can arrive on the primed DNA strand, namely, PCNA arrives first, Pol δ arrives first, or they both arrive simultaneously. Bottom: departure of Pol δ or PCNA could occur in one of the three ways, namely, Pol δ could depart first, PCNA could depart first, or the two could leave simultaneously. (B) An example of an image series of a typical colocalization binding event observed by dual-color SMT. In this particular event, colocalization occurred from frames 3–7 (~6-s duration). Note the Halo-PCNA signal appears before the SNAP-Pol δ signal and disappears after the Pol δ signal. Scale bars represent 500 nm. (C) Histogram of the arrival and departure order of SNAP-Pol δ and Halo-PCNA molecules during observed genomic colocalization events. The percentage of SNAP-Pol δ and Halo-PCNA molecules arriving and departing first or at the same time are indicated. Error bars indicate SD, as determined by Bootstrap analysis (1,000 iterations) of observed colocalizations. The predominant order of events observed is that PCNA loading precedes Pol δ binding, followed by Pol δ dissociation preceding PCNA unloading. (D) Scatterplot of SNAP-Pol δ arrival (binding) and departure (dissociation) relative to Halo-PCNA binding and dissociation during nuclear colocalization events. Timelines (blue and orange bars) shown in each quadrant indicate the binding order of SNAP-Pol δ and Halo-PCNA and time interval between arrival and departure of Pol δ/PCNA for colocalizations within that quadrant (SNAP-Pol δ arriving/departing before/ after Halo-PCNA). Blue bars indicate the presence of genome-bound Halo-PCNA during colocalization events; orange bars indicate the presence of genome-bound SNAP-Pol δ (p125) during colocalization events. Median time intervals (s) between arrival (before colocalization) and departure (after colocalization) of PCNA/Pol δ are shown below bars; median time interval (s) of colocalization shown above bars. The 95% confidence interval of median times is reported in Figure S2 . Percentage of total colocalizations represented by the specific order of assembly/disassembly in each quadrant is indicated under timelines. Plots reveal multiple assembly/disassembly pathways of the holoenzyme. A total of 270 colocalization events from 59 nuclei of LOX C11 cells from 4 independent experiments were analyzed.

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Binding Assay, Sequencing

    SMT in Live Cells by High-Resolution Microscopy Reveals Dynamic Interactions between Pol δ and PCNA That Are PIP Box Mediated (A) Schematic detailing live-cell SMT of fluorescently tagged Pol δ (p125) and PCNA. (i) Labeled cells in an imaging dish are illuminated by a laser focused to produce a narrow layer of illumination across the focal plane (HILO illumination); only labeled proteins within the focal plane are excited and fluoresce. The 2,000-frame time-lapse photomicrographic videos are captured while a high speed filter continuously alternates between SNAP and Halo dye channels to obtain near-simultaneous imaging of labeled SNAP- and Halo-tagged proteins. (ii) An image series of a single binding event. Frames have been skipped for simplicity; this event lasted from frame 3 s to 10.5 s. (iii) All of the signals for a given labeled protein seen within a nucleus (outlined) throughout an entire video capture are localized in a 2D map. Individual signals in consecutive frames located within a highly confined area (based on expected diffusion constants) are linked together to create a track, which identifies a binding event. The average position of the binding event (mean x and y-position) is indicated by a red X. Spatiotemporal information that defines a track can be used to classify binding events as short lived (

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: SMT in Live Cells by High-Resolution Microscopy Reveals Dynamic Interactions between Pol δ and PCNA That Are PIP Box Mediated (A) Schematic detailing live-cell SMT of fluorescently tagged Pol δ (p125) and PCNA. (i) Labeled cells in an imaging dish are illuminated by a laser focused to produce a narrow layer of illumination across the focal plane (HILO illumination); only labeled proteins within the focal plane are excited and fluoresce. The 2,000-frame time-lapse photomicrographic videos are captured while a high speed filter continuously alternates between SNAP and Halo dye channels to obtain near-simultaneous imaging of labeled SNAP- and Halo-tagged proteins. (ii) An image series of a single binding event. Frames have been skipped for simplicity; this event lasted from frame 3 s to 10.5 s. (iii) All of the signals for a given labeled protein seen within a nucleus (outlined) throughout an entire video capture are localized in a 2D map. Individual signals in consecutive frames located within a highly confined area (based on expected diffusion constants) are linked together to create a track, which identifies a binding event. The average position of the binding event (mean x and y-position) is indicated by a red X. Spatiotemporal information that defines a track can be used to classify binding events as short lived (

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Microscopy, Labeling, Imaging, Binding Assay, Diffusion-based Assay

    Pol δ Forms Foci of Binding Events That Are Spatiotemporally Related (A) Discontinuous lagging strand replication involves cycles of Pol δ binding synthesis release and Pol δ revisiting. (B) Global 2D map of WT Pol δ nuclear binding events shown at left. Chromatin binding events lasting longer than 1.7 s are shown (red dots). Clustering analysis algorithms revealed repeated Pol δ binding events within small foci (hubs) shaded in blue. Expanded inset (bottom right) of boxed region in left panel illustrates clustering of Pol δ binding events (blue dots) within hubs shaded in yellow (with black outline). (C) Close-up image series of a single hub (yellow box) during cycles of revisiting by WT Pol δ shown in top panel. A playback timeline of binding events is shown below image series. Black bars indicate bound Pol δ. Bar width corresponds to duration of binding event (when Pol δ appears then disappears). (D) Comparison of the number of Pol δ hubs per 1,000 binding events for SNAP-Pol δ WT or SNAP-Pol δ Dead. (E) Comparison of the number of Pol δ binding events per hub for SNAP-Pol δ WT or SNAP-Pol δ Dead (****p = 0.001). Binding events from 59 nuclei of SNAP-Pol δ WT (C11) cells from 4 independent experiments and 13 nuclei of SNAP-Pol δ Dead (C2) cells from 2 independent experiments were analyzed. A Kruskal-Wallis test was used to determine pairwise significance.

    Journal: Cell reports

    Article Title: Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo

    doi: 10.1016/j.celrep.2019.12.101

    Figure Lengend Snippet: Pol δ Forms Foci of Binding Events That Are Spatiotemporally Related (A) Discontinuous lagging strand replication involves cycles of Pol δ binding synthesis release and Pol δ revisiting. (B) Global 2D map of WT Pol δ nuclear binding events shown at left. Chromatin binding events lasting longer than 1.7 s are shown (red dots). Clustering analysis algorithms revealed repeated Pol δ binding events within small foci (hubs) shaded in blue. Expanded inset (bottom right) of boxed region in left panel illustrates clustering of Pol δ binding events (blue dots) within hubs shaded in yellow (with black outline). (C) Close-up image series of a single hub (yellow box) during cycles of revisiting by WT Pol δ shown in top panel. A playback timeline of binding events is shown below image series. Black bars indicate bound Pol δ. Bar width corresponds to duration of binding event (when Pol δ appears then disappears). (D) Comparison of the number of Pol δ hubs per 1,000 binding events for SNAP-Pol δ WT or SNAP-Pol δ Dead. (E) Comparison of the number of Pol δ binding events per hub for SNAP-Pol δ WT or SNAP-Pol δ Dead (****p = 0.001). Binding events from 59 nuclei of SNAP-Pol δ WT (C11) cells from 4 independent experiments and 13 nuclei of SNAP-Pol δ Dead (C2) cells from 2 independent experiments were analyzed. A Kruskal-Wallis test was used to determine pairwise significance.

    Article Snippet: Primary antibodies used were: anti-human DNA Pol δ p125 (mouse monoclonal, Santa Cruz), anti-human PCNA (mouse monoclonal, Abcam), anti-SNAP tag (rabbit polyclonal, New England Biolabs), anti-Halo tag (mouse monoclonal, Promega), anti-α-Tubulin (mouse monoclonal, Sigma), and anti-actin (rabbit polyclonal, Sigma).

    Techniques: Binding Assay

    Validation of Mis12-LoKI platforms. ( A ) Schematic depicting the Mis12-LoKI viral construct with mCherry-SNAP-Mis12 under the control of a doxycycline-inducible promoter. ( B ) Immunoblot of SNAP-Mis12 (top) expression at selected time points after removal of doxycycline and GAPDH loading controls (bottom). Quantification of amalgamated data is presented below. ( C ) Pulse-chase experiments measuring CLP-MLN8237’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). Experiments were conducted at least two times (N = 2–3). Data are mean ± s.e.m. Source files for analysis of pulse-chase experiments are available in Figure 6—figure supplement 1—source data 1 . Analysis for pulse-chase experiments with CLP-BI2536 in SNAP-Mis12 cells.

    Journal: eLife

    Article Title: Subcellular drug targeting illuminates local kinase action

    doi: 10.7554/eLife.52220

    Figure Lengend Snippet: Validation of Mis12-LoKI platforms. ( A ) Schematic depicting the Mis12-LoKI viral construct with mCherry-SNAP-Mis12 under the control of a doxycycline-inducible promoter. ( B ) Immunoblot of SNAP-Mis12 (top) expression at selected time points after removal of doxycycline and GAPDH loading controls (bottom). Quantification of amalgamated data is presented below. ( C ) Pulse-chase experiments measuring CLP-MLN8237’s ability to block CLP-rhodamine conjugation to LoKI-on. In-gel rhodamine fluorescence (top), immunoblot of SNAP loading controls (mid), and fluorescence quantification of pulse-chase experiments (bottom). Experiments were conducted at least two times (N = 2–3). Data are mean ± s.e.m. Source files for analysis of pulse-chase experiments are available in Figure 6—figure supplement 1—source data 1 . Analysis for pulse-chase experiments with CLP-BI2536 in SNAP-Mis12 cells.

    Article Snippet: Proteins were transferred to nitrocellulose for immunoblotting and probed with Anti-SNAP-tag rabbit antibody (NEB) and Anti-GAPDH−HRP mouse mAb, (Novus).

    Techniques: Construct, Expressing, Pulse Chase, Blocking Assay, Conjugation Assay, Fluorescence

    Characterization of Plk1 inhibition with CLP-BI2536. ( A ) Immunofluorescence detection of pT210-Plk1 as an index of kinase activity in parental U2OS cells treated with DMSO or unconjugated BI2536 for 4 hr. ( B ) Quantification of centrosomal pT210-Plk1 immunofluorescence collected from parental U2OS cells. ( C ) Quantification of total Plk1 immunofluorescence at centrosomes in LoKI-expressing cells after 4 hr CLP-BI2536 treatment; 250 nM, LoKI-off, n = 55, LoKI-on, n = 47, *p=0.0217; 500 nM, LoKI-off, n = 52, LoKI-on, n = 51, *p=0.0295. ( D ) Quantification of pT210-Plk1 immunofluorescence in control cells lacking SNAP expression (not induced with doxycycline) after 4 hr CLP-BI2536 treatment. ( E ) Immunoblot detection of pT210-Plk1 (blot 2) and pT288-AurA (blot 4) in LoKI-off and LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO, 250 nM CLP-BI2536, or 100 nM CLP-MLN8237. Total Plk1 (blot 3), AurA (blot 5), SNAP-PACT (blot 1), and GAPDH (blot 6) are also depicted. ( F ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr in RPE and HeLa cells and GAPDH loading controls (bottom). ( G–J ) Immunofluorescence detection of pT210-Plk1 at centrosomes in LoKI-off (top) and LoKI-on (bottom) RPE ( G ) and HeLa ( I ) cells treated with 250 nM CLP-BI2536 for 4 hr. Quantification of pT210-Plk1 immunofluorescence at centrosomes in RPE ( H ) 250 nM, LoKI-off, n = 70, LoKI-on, n = 47, ****p

    Journal: eLife

    Article Title: Subcellular drug targeting illuminates local kinase action

    doi: 10.7554/eLife.52220

    Figure Lengend Snippet: Characterization of Plk1 inhibition with CLP-BI2536. ( A ) Immunofluorescence detection of pT210-Plk1 as an index of kinase activity in parental U2OS cells treated with DMSO or unconjugated BI2536 for 4 hr. ( B ) Quantification of centrosomal pT210-Plk1 immunofluorescence collected from parental U2OS cells. ( C ) Quantification of total Plk1 immunofluorescence at centrosomes in LoKI-expressing cells after 4 hr CLP-BI2536 treatment; 250 nM, LoKI-off, n = 55, LoKI-on, n = 47, *p=0.0217; 500 nM, LoKI-off, n = 52, LoKI-on, n = 51, *p=0.0295. ( D ) Quantification of pT210-Plk1 immunofluorescence in control cells lacking SNAP expression (not induced with doxycycline) after 4 hr CLP-BI2536 treatment. ( E ) Immunoblot detection of pT210-Plk1 (blot 2) and pT288-AurA (blot 4) in LoKI-off and LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO, 250 nM CLP-BI2536, or 100 nM CLP-MLN8237. Total Plk1 (blot 3), AurA (blot 5), SNAP-PACT (blot 1), and GAPDH (blot 6) are also depicted. ( F ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr in RPE and HeLa cells and GAPDH loading controls (bottom). ( G–J ) Immunofluorescence detection of pT210-Plk1 at centrosomes in LoKI-off (top) and LoKI-on (bottom) RPE ( G ) and HeLa ( I ) cells treated with 250 nM CLP-BI2536 for 4 hr. Quantification of pT210-Plk1 immunofluorescence at centrosomes in RPE ( H ) 250 nM, LoKI-off, n = 70, LoKI-on, n = 47, ****p

    Article Snippet: Proteins were transferred to nitrocellulose for immunoblotting and probed with Anti-SNAP-tag rabbit antibody (NEB) and Anti-GAPDH−HRP mouse mAb, (Novus).

    Techniques: Inhibition, Immunofluorescence, Activity Assay, Expressing

    Validation of the LoKI system. ( A ) Full chemical structure of CLP-BI2536. ( B ) Dose-response curve depicting in vitro Plk1 inhibition with increasing concentrations of CLP-BI2536 conjugated to purified SNAP. ( C ) Schematic of LoKI viral construct with mCherry-SNAP-PACT under control of a doxycycline-inducible promoter. ( D ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr and GAPDH loading controls (bottom). ( E ) Immunoblot of SNAP-PACT (top) expression at selected time points after removal of doxycycline and GAPDH loading controls (bottom). Quantification of amalgamated data is presented below. ( F ) Immunofluorescent detection of interphase (top) and mitotic (bottom) U2OS cells showing α-tubulin (left and green), DNA (mid and blue), and SNAP (right and magenta). ( G, H ) Diagram of centrosomal LoKI-on ( G ) platform with drugs conjugated and LoKI-off ( H ) platform containing a mutation that occludes CLP binding. Experiments were conducted at least two times (N = 2–3). Data are mean ± s.e.m.

    Journal: eLife

    Article Title: Subcellular drug targeting illuminates local kinase action

    doi: 10.7554/eLife.52220

    Figure Lengend Snippet: Validation of the LoKI system. ( A ) Full chemical structure of CLP-BI2536. ( B ) Dose-response curve depicting in vitro Plk1 inhibition with increasing concentrations of CLP-BI2536 conjugated to purified SNAP. ( C ) Schematic of LoKI viral construct with mCherry-SNAP-PACT under control of a doxycycline-inducible promoter. ( D ) Immunoblot confirming SNAP-PACT (top) expression after induction with doxycycline for 72 hr and GAPDH loading controls (bottom). ( E ) Immunoblot of SNAP-PACT (top) expression at selected time points after removal of doxycycline and GAPDH loading controls (bottom). Quantification of amalgamated data is presented below. ( F ) Immunofluorescent detection of interphase (top) and mitotic (bottom) U2OS cells showing α-tubulin (left and green), DNA (mid and blue), and SNAP (right and magenta). ( G, H ) Diagram of centrosomal LoKI-on ( G ) platform with drugs conjugated and LoKI-off ( H ) platform containing a mutation that occludes CLP binding. Experiments were conducted at least two times (N = 2–3). Data are mean ± s.e.m.

    Article Snippet: Proteins were transferred to nitrocellulose for immunoblotting and probed with Anti-SNAP-tag rabbit antibody (NEB) and Anti-GAPDH−HRP mouse mAb, (Novus).

    Techniques: In Vitro, Inhibition, Purification, Construct, Expressing, Mutagenesis, Binding Assay

    Characterization of AurA inhibition with CLP-MLN8237. ( A ) Quantification of pS69-Hec1 immunofluorescence at centrosomes after 4 hr CLP-MLN8237 treatment. ( B ) Immunoblot detection of pS69-Hec1 (blot 2) and pT288-AurA (blot 3) in Mis12-LoKI-off and Mis12-LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO or 100 nM CLP-MLN8237. Total AurA (blot 3), SNAP-PACT (blot 1), and GAPDH (blot 5) are also depicted. ( C ) Quantification of pS69-Hec1 immunofluorescence at kinetochores after 4 hr CLP-MLN8237 treatment in SNAP-PACT expressing U2OS cells. Points in ( A ) and ( C ) represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant.

    Journal: eLife

    Article Title: Subcellular drug targeting illuminates local kinase action

    doi: 10.7554/eLife.52220

    Figure Lengend Snippet: Characterization of AurA inhibition with CLP-MLN8237. ( A ) Quantification of pS69-Hec1 immunofluorescence at centrosomes after 4 hr CLP-MLN8237 treatment. ( B ) Immunoblot detection of pS69-Hec1 (blot 2) and pT288-AurA (blot 3) in Mis12-LoKI-off and Mis12-LoKI-on expressing cells collected via mitotic shake-off. Cells were treated for 16 hr with nocodazole and 4 hr with nocodazole plus DMSO or 100 nM CLP-MLN8237. Total AurA (blot 3), SNAP-PACT (blot 1), and GAPDH (blot 5) are also depicted. ( C ) Quantification of pS69-Hec1 immunofluorescence at kinetochores after 4 hr CLP-MLN8237 treatment in SNAP-PACT expressing U2OS cells. Points in ( A ) and ( C ) represent individual cells ( n ). Data normalized to DMSO. Experiments were conducted at least three times (N = 3) and P values were calculated by unpaired two-tailed Student’s t-test. Data are mean ± s.e.m. NS, not significant.

    Article Snippet: Proteins were transferred to nitrocellulose for immunoblotting and probed with Anti-SNAP-tag rabbit antibody (NEB) and Anti-GAPDH−HRP mouse mAb, (Novus).

    Techniques: Inhibition, Immunofluorescence, Expressing, Two Tailed Test