Structured Review

BPS Bioscience histone h4
Cytotoxic effects of extracellular <t>histones</t> on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)
Histone H4, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury"

Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury

Journal: The FASEB Journal

doi: 10.1096/fj.13-236380

Cytotoxic effects of extracellular histones on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)
Figure Legend Snippet: Cytotoxic effects of extracellular histones on alveolar epithelial cells. A ) LDH release from LA-4 and MLE-12 lung alveolar epithelial cells exposed to purified histones (50 μg/ml, 1 h), chromogenic assay. B ) [Ca 2+ ] i staining in untreated (Ctrl)

Techniques Used: Purification, Chromogenic Assay, Staining

Administration of extracellular histones into airways induces severe disturbances in alveolar-capillary gas exchange. Purified histones (50 μg/g body weight) were administered i.t. to Sprague-Dawley rats with implanted carotid artery catheters.
Figure Legend Snippet: Administration of extracellular histones into airways induces severe disturbances in alveolar-capillary gas exchange. Purified histones (50 μg/g body weight) were administered i.t. to Sprague-Dawley rats with implanted carotid artery catheters.

Techniques Used: Purification

Protective effects of neutralization of extracellular histones during ALI. A ) Reduced alveolar permeability disturbances after C5a-ALI with treatment of neutralizing anti-H4 antibody (BWA3, 250 μg i.v. + 50 μg i.t.) or isotype control
Figure Legend Snippet: Protective effects of neutralization of extracellular histones during ALI. A ) Reduced alveolar permeability disturbances after C5a-ALI with treatment of neutralizing anti-H4 antibody (BWA3, 250 μg i.v. + 50 μg i.t.) or isotype control

Techniques Used: Neutralization, Permeability

Detection of extracellular histones during ALI in humans or mice. A ) Top panel: Western blotting of histone H4 in BALF from 2 humans with ALI/ARDS (40589; 50131) at sequential time points (4–21 d), a healthy volunteer (Ctrl BALF, negative control),
Figure Legend Snippet: Detection of extracellular histones during ALI in humans or mice. A ) Top panel: Western blotting of histone H4 in BALF from 2 humans with ALI/ARDS (40589; 50131) at sequential time points (4–21 d), a healthy volunteer (Ctrl BALF, negative control),

Techniques Used: Mouse Assay, Western Blot, Negative Control

Induction of acute inflammation in lungs by extracellular histones. A ) Disruption of alveolar permeability in C57BL/6J mice after intratracheal deposition of purified histones at different doses, 8 h; BALF albumin ELISA. B ) Time course for alveolar permeability
Figure Legend Snippet: Induction of acute inflammation in lungs by extracellular histones. A ) Disruption of alveolar permeability in C57BL/6J mice after intratracheal deposition of purified histones at different doses, 8 h; BALF albumin ELISA. B ) Time course for alveolar permeability

Techniques Used: Permeability, Mouse Assay, Purification, Enzyme-linked Immunosorbent Assay

Extracellular histones mediate lung consolidation and acute histopathology in lungs. A ) High-resolution MRI of lungs from C57BL/6J mice after PBS i.t. (sham treatment) or purified histones (20 μg/g body weight), 6 h. Arrowheads indicate signal-intense
Figure Legend Snippet: Extracellular histones mediate lung consolidation and acute histopathology in lungs. A ) High-resolution MRI of lungs from C57BL/6J mice after PBS i.t. (sham treatment) or purified histones (20 μg/g body weight), 6 h. Arrowheads indicate signal-intense

Techniques Used: Histopathology, Magnetic Resonance Imaging, Mouse Assay, Purification

Whole-body plethysmography following airway administration of histones. Sprague-Dawley rats received purified histones (50 μg/g body weight i.t.) or PBS i.t (sham treatment). Respiration was monitored before the intervention and at intervals during
Figure Legend Snippet: Whole-body plethysmography following airway administration of histones. Sprague-Dawley rats received purified histones (50 μg/g body weight i.t.) or PBS i.t (sham treatment). Respiration was monitored before the intervention and at intervals during

Techniques Used: Purification

Related Articles

Incubation:

Article Title: Identification and Characterization of Propionylation at Histone H3 Lysine 23 in Mammalian Cells *
Article Snippet: .. For the depropionylation assay, histone H3 was first incubated with p300 in the presence of [14 C]propionyl-CoA (or [14 C]acetyl-CoA as a control) for 1 h, and the reaction was stopped by heating at 60 °C for 5 min. 10 μl (100 μg/300 μl of solution) of human Sir2 proteins (sirtuins 1–3; BPS Bioscience) was then added to the solution together with 50 m m NAD+ . .. Samples were then subjected to SDS-PAGE analysis with Coomassie Blue staining and autoradiography.

Purification:

Article Title: Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury
Article Snippet: .. The following reagents were used: anti-Ly6G antibody (clone RB6-8C5) and rat IgG2bκ isotype antibody (eBioscience), neutralizing anti-histone H4 antibody (clone BWA3; ref. ) and mouse IgG1κ isotype antibody (BioLegend), rmC5a (R & D Systems), purified histones from calf thymus (Sigma), histone H1 and histone H3 (Roche), and histone H4 (BPS Bioscience, San Diego, CA, USA). .. The lactate dehydrogenase (LDH) assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to instructions by the manufacturer.

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    BPS Bioscience recombinant hdac1
    Exifone’s mechanism of action with <t>HDAC1</t> is consistent with mixed non-essential activation. ( A ) Reaction mechanism of HDAC1 activation by exifone as determined by varying concentrations of both substrate and activator. Increasing exifone concentration decreased the apparent substrate K m for Bio-H4K12Ac peptide (B) and increased the V max app ( C ). Replot of slope (ratio of K m / V max ) vs. exifone concentration shown as descending hyperbolic curve ( D ).
    Recombinant Hdac1, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BPS Bioscience tppp p25
    Effect of SIRT2 on the <t>TPPP/p25-induced</t> tubulin polymerization. ( a ) Tubulin polymerization (6 μM) promoted by TPPP/p25 (3 μM) in the absence and presence of inactive or active (with 500 μM NAD + ) SIRT2 (25 μM). The tubulin polymerization was induced by the addition of TPPP/p25 or TPPP/p25 preincubated with SIRT2. ( b ) Electron microscopic images of the pellet fractions produced by TPPP/p25-induced tubulin assembly in absence and presence of SIRT2 (with NAD + ) ( a ). ( c ) and ( d ) Western blot images of supernatant (S) and pellet (P) fractions using acetyl-tubulin antibody, when polymerization was induced by TPPP/p25 premixed with SIRT2 ( c ) or SIRT2 was added to the TPPP/p25-assembled tubulin ( d ). All data of the representative experiments can be found in the source data file. The images of the full-length blots are presented in Supplementary Figs S2 and S3 .
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    BPS Bioscience g9a hmt
    Histogram of <t>HMT</t> <t>G9a</t> primary screening results
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    BPS Bioscience hdac 6
    Percent enzyme activity remaining for HDAC-1 (top, 0.25 μM) and <t>HDAC-6</t> (bottom, 0.16 μM) in the presence of 53 or 22 nM SAHA (IC50 = 53 nM and 22 nM, respectively) and increasing concentrations of MT. The ratios of MPi to MT are indicated
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    Image Search Results


    Exifone’s mechanism of action with HDAC1 is consistent with mixed non-essential activation. ( A ) Reaction mechanism of HDAC1 activation by exifone as determined by varying concentrations of both substrate and activator. Increasing exifone concentration decreased the apparent substrate K m for Bio-H4K12Ac peptide (B) and increased the V max app ( C ). Replot of slope (ratio of K m / V max ) vs. exifone concentration shown as descending hyperbolic curve ( D ).

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Exifone’s mechanism of action with HDAC1 is consistent with mixed non-essential activation. ( A ) Reaction mechanism of HDAC1 activation by exifone as determined by varying concentrations of both substrate and activator. Increasing exifone concentration decreased the apparent substrate K m for Bio-H4K12Ac peptide (B) and increased the V max app ( C ). Replot of slope (ratio of K m / V max ) vs. exifone concentration shown as descending hyperbolic curve ( D ).

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Activation Assay, Concentration Assay

    Exifone as an HDAC1 activator increases histone deacetylation in human neural progenitor cells (NPCs). NPCs were treated with various concentrations of exifone for 6h or 18h, followed by fixation and immunostaining for H3K9Ac in nuclei. ( A ) Representative images of H3K9Ac immunostaining of iPSC-derived NPCs, showing dimmer nuclei in 0.5 μM and 2 μM exifone-treated (6h) cultures. Quantitative image analysis was performed to determine the intensity of H3K9Ac in the nuclei. ( B ) Quantitation of the H3K9Ac intensity levels indicates effective activation of HDAC(s) in NPCs, thereby leading to decreased H3K9Ac levels in 6h or 18h treated cultures. Error bars represent standard error of the mean (SEM) based on N=3 biological replicates per treatment. Mean pixel intensities were collected from 9 images per biological replicate totaling approximately 3,500 nuclei. Unpaired t test: **** p

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Exifone as an HDAC1 activator increases histone deacetylation in human neural progenitor cells (NPCs). NPCs were treated with various concentrations of exifone for 6h or 18h, followed by fixation and immunostaining for H3K9Ac in nuclei. ( A ) Representative images of H3K9Ac immunostaining of iPSC-derived NPCs, showing dimmer nuclei in 0.5 μM and 2 μM exifone-treated (6h) cultures. Quantitative image analysis was performed to determine the intensity of H3K9Ac in the nuclei. ( B ) Quantitation of the H3K9Ac intensity levels indicates effective activation of HDAC(s) in NPCs, thereby leading to decreased H3K9Ac levels in 6h or 18h treated cultures. Error bars represent standard error of the mean (SEM) based on N=3 biological replicates per treatment. Mean pixel intensities were collected from 9 images per biological replicate totaling approximately 3,500 nuclei. Unpaired t test: **** p

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Immunostaining, Derivative Assay, Quantitation Assay, Activation Assay

    Biolayer interferometry-based biophysical assays of exifone binding to HDAC1. ( A and B ) Exifone directly binds HDAC1 as measured using BLI whereas the structurally simpler analog, benzophenone does not exhibit a binding interaction. ( C ). The processed data graphs were obtained after reference (n=3) subtraction and show the acquisition of real-time data in a binding kinetics experiment. After an initial baseline step in the assay buffer, streptavidin biosensors were dipped into solution with the biotinylated HDAC1 for the loading step. Subsequently, a second baseline was performed, followed by association and dissociation of the small molecule analyte (n=5) in solution. Successful binding interaction results in distinct spectral shift pattern that is represented on the sensorgram as a change in the wavelength (nm shift). The data were fit globally with the 1:1 binding model and a representative plot of R eq vs. exifone concentration for the estimation of K D is shown ( B ).

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Biolayer interferometry-based biophysical assays of exifone binding to HDAC1. ( A and B ) Exifone directly binds HDAC1 as measured using BLI whereas the structurally simpler analog, benzophenone does not exhibit a binding interaction. ( C ). The processed data graphs were obtained after reference (n=3) subtraction and show the acquisition of real-time data in a binding kinetics experiment. After an initial baseline step in the assay buffer, streptavidin biosensors were dipped into solution with the biotinylated HDAC1 for the loading step. Subsequently, a second baseline was performed, followed by association and dissociation of the small molecule analyte (n=5) in solution. Successful binding interaction results in distinct spectral shift pattern that is represented on the sensorgram as a change in the wavelength (nm shift). The data were fit globally with the 1:1 binding model and a representative plot of R eq vs. exifone concentration for the estimation of K D is shown ( B ).

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Binding Assay, Concentration Assay

    Exifone is capable of reducing inhibition caused by pre-incubation of HDAC1 with active site inhibitor CI-994. ( A ) Concentration response curves for the HDAC inhibitor CI-994 in the absence and presence of the HDAC activator exifone. CI-994 shows an IC50 value of 0.037 μM and the top fitted value is less than 100%. Preincubation of HDAC1 for 15 minutes with 1 μM or 10 μM exifone resulted in the top values for % deacetylase activity reaching up to 620% and 450% respectively. The results indicate that preincubation of exifone can still result in significant HDAC1 activation at lower concentrations of CI-994. ( B ) A dose-dependent increase in HDAC1 activity was observed even if HDAC1 was pre-incubated for two hours with 1 μM CI-994, 5 μM CI-994, 10 μM CI-994, as shown by the best-fit values.

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Exifone is capable of reducing inhibition caused by pre-incubation of HDAC1 with active site inhibitor CI-994. ( A ) Concentration response curves for the HDAC inhibitor CI-994 in the absence and presence of the HDAC activator exifone. CI-994 shows an IC50 value of 0.037 μM and the top fitted value is less than 100%. Preincubation of HDAC1 for 15 minutes with 1 μM or 10 μM exifone resulted in the top values for % deacetylase activity reaching up to 620% and 450% respectively. The results indicate that preincubation of exifone can still result in significant HDAC1 activation at lower concentrations of CI-994. ( B ) A dose-dependent increase in HDAC1 activity was observed even if HDAC1 was pre-incubated for two hours with 1 μM CI-994, 5 μM CI-994, 10 μM CI-994, as shown by the best-fit values.

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Inhibition, Incubation, Concentration Assay, Histone Deacetylase Assay, Activity Assay, Activation Assay

    Exifone is at least four-fold selective for activating HDAC1 when compared with HDAC2. The data were fitted with the equation for sigmoidal doseresponse (variable slope) to estimate the EC 50 values for exifone, and the best fit values are shown as calculated using the RapidFire mass spectrometry assay. The observed EC 50 values under the defined conditions for HDAC1 are 0.02 μM as compared with 0.082 μM for HDAC2. Estimated EC 1.5 values for HDAC1 and HDAC2 for this set of data were 0.002 μM and 0.015 μM respectively.

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Exifone is at least four-fold selective for activating HDAC1 when compared with HDAC2. The data were fitted with the equation for sigmoidal doseresponse (variable slope) to estimate the EC 50 values for exifone, and the best fit values are shown as calculated using the RapidFire mass spectrometry assay. The observed EC 50 values under the defined conditions for HDAC1 are 0.02 μM as compared with 0.082 μM for HDAC2. Estimated EC 1.5 values for HDAC1 and HDAC2 for this set of data were 0.002 μM and 0.015 μM respectively.

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Mass Spectrometry

    Exifone as a potent small molecule activator of HDAC1. Structure of exifone ( A ) and benzophenone ( B ), respectively. Dose-dependent activation of HDAC1 by exifone in RapidFire mass spectrometry assay for HDAC1 with 1 μM Bio-H4K12Ac substrate ( C ) or 1 μM Bio-p53K382Ac substrate ( E ). The compound benzophenone ( B ), a structurally simpler compound that lacks the six hydroxyl groups does not cause HDAC1 activation ( D and F ). (G) Exifone increased the rate of deacetylation reaction by HDAC1 as observed via monitoring the percentage of substrate conversion. Mass spectrometry-based (Rapid-Fire) HDAC1 deacetylase activity assay was performed with 1 μM Bio-H4K12Ac substrate and 40 nM enzyme. The figure demonstrates the linearity of reaction progression as a function of time in the absence (control) and the presence of exifone. (H) Dose-dependent activation of HDAC1 by exifone at variable concentrations of the acetylated substrate peptide (Bio-H4K12Ac). The highest level of enzyme activation was observed when the substrate concentration is significantly lower than the apparent K m value for the acetylated peptide substrate ([S]

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Exifone as a potent small molecule activator of HDAC1. Structure of exifone ( A ) and benzophenone ( B ), respectively. Dose-dependent activation of HDAC1 by exifone in RapidFire mass spectrometry assay for HDAC1 with 1 μM Bio-H4K12Ac substrate ( C ) or 1 μM Bio-p53K382Ac substrate ( E ). The compound benzophenone ( B ), a structurally simpler compound that lacks the six hydroxyl groups does not cause HDAC1 activation ( D and F ). (G) Exifone increased the rate of deacetylation reaction by HDAC1 as observed via monitoring the percentage of substrate conversion. Mass spectrometry-based (Rapid-Fire) HDAC1 deacetylase activity assay was performed with 1 μM Bio-H4K12Ac substrate and 40 nM enzyme. The figure demonstrates the linearity of reaction progression as a function of time in the absence (control) and the presence of exifone. (H) Dose-dependent activation of HDAC1 by exifone at variable concentrations of the acetylated substrate peptide (Bio-H4K12Ac). The highest level of enzyme activation was observed when the substrate concentration is significantly lower than the apparent K m value for the acetylated peptide substrate ([S]

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques: Activation Assay, Mass Spectrometry, Histone Deacetylase Assay, Activity Assay, Concentration Assay

    Selectivity profiling of exifone against neurodegeneration relevant targets. Kinetic parameters estimated via BLI assays indicate exifone shows a preference interaction with HDAC1. Exifone has the highest affinity for HDAC1 when compared with HDAC2, HDAC8, and CDK5/p25 as evidenced by ( A ) the lowest value of equilibrium dissociation constant, and ( B ) the highest value for the association rate constant ( k a ) that represents the number of complexes formed per second between the small molecule and the biotinylated protein in a 1 Molar solution.

    Journal: bioRxiv

    Article Title: Exifone is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration

    doi: 10.1101/2020.03.02.973636

    Figure Lengend Snippet: Selectivity profiling of exifone against neurodegeneration relevant targets. Kinetic parameters estimated via BLI assays indicate exifone shows a preference interaction with HDAC1. Exifone has the highest affinity for HDAC1 when compared with HDAC2, HDAC8, and CDK5/p25 as evidenced by ( A ) the lowest value of equilibrium dissociation constant, and ( B ) the highest value for the association rate constant ( k a ) that represents the number of complexes formed per second between the small molecule and the biotinylated protein in a 1 Molar solution.

    Article Snippet: Recombinant HDAC1 and HDAC2 enzyme preps were from BPS Biosciences (San Diego, CA).

    Techniques:

    Effect of SIRT2 on the TPPP/p25-induced tubulin polymerization. ( a ) Tubulin polymerization (6 μM) promoted by TPPP/p25 (3 μM) in the absence and presence of inactive or active (with 500 μM NAD + ) SIRT2 (25 μM). The tubulin polymerization was induced by the addition of TPPP/p25 or TPPP/p25 preincubated with SIRT2. ( b ) Electron microscopic images of the pellet fractions produced by TPPP/p25-induced tubulin assembly in absence and presence of SIRT2 (with NAD + ) ( a ). ( c ) and ( d ) Western blot images of supernatant (S) and pellet (P) fractions using acetyl-tubulin antibody, when polymerization was induced by TPPP/p25 premixed with SIRT2 ( c ) or SIRT2 was added to the TPPP/p25-assembled tubulin ( d ). All data of the representative experiments can be found in the source data file. The images of the full-length blots are presented in Supplementary Figs S2 and S3 .

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Effect of SIRT2 on the TPPP/p25-induced tubulin polymerization. ( a ) Tubulin polymerization (6 μM) promoted by TPPP/p25 (3 μM) in the absence and presence of inactive or active (with 500 μM NAD + ) SIRT2 (25 μM). The tubulin polymerization was induced by the addition of TPPP/p25 or TPPP/p25 preincubated with SIRT2. ( b ) Electron microscopic images of the pellet fractions produced by TPPP/p25-induced tubulin assembly in absence and presence of SIRT2 (with NAD + ) ( a ). ( c ) and ( d ) Western blot images of supernatant (S) and pellet (P) fractions using acetyl-tubulin antibody, when polymerization was induced by TPPP/p25 premixed with SIRT2 ( c ) or SIRT2 was added to the TPPP/p25-assembled tubulin ( d ). All data of the representative experiments can be found in the source data file. The images of the full-length blots are presented in Supplementary Figs S2 and S3 .

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Produced, Western Blot

    Quantification of the pair-wise interactions of SIRT2, TPPP/p25 and tubulin by ELISA. ( a ) Saturation curves of TPPP/p25 (●) and tubulin (○), when the plate was coated with SIRT2; and that of tubulin (▲), when the plate was coated with TPPP/p25. Concentrations of the coated proteins were 5 μg/ml. The data are presented as mean ± SD, n = 3 for TPPP/p25-SIRT2, n = 4 for tubulin-SIRT2, and n = 11 for TPPP/p25-tubulin. ( b ) Displacement of 125 nM TPPP/p25 from the coated SIRT2 by tubulin added at various concentrations detected by TPPP/p25 antibody. The data are presented as mean ± SD, n = 3. ( c ) Binding of TPPP/p25 (250 nM) and tubulin (1 μM) to the immobilized SIRT2 as detected by TPPP/p25 and tubulin antibodies, respectively. The data are presented as mean ± SD, n = 4. *p = 2.61E-04 when TPPP/p25 with tubulin compared to TPPP/p25 alone, and *p = 1.76E-02 when tubulin with TPPP/p25 compared to tubulin alone (two-sided, unpaired Student’s t-test).

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Quantification of the pair-wise interactions of SIRT2, TPPP/p25 and tubulin by ELISA. ( a ) Saturation curves of TPPP/p25 (●) and tubulin (○), when the plate was coated with SIRT2; and that of tubulin (▲), when the plate was coated with TPPP/p25. Concentrations of the coated proteins were 5 μg/ml. The data are presented as mean ± SD, n = 3 for TPPP/p25-SIRT2, n = 4 for tubulin-SIRT2, and n = 11 for TPPP/p25-tubulin. ( b ) Displacement of 125 nM TPPP/p25 from the coated SIRT2 by tubulin added at various concentrations detected by TPPP/p25 antibody. The data are presented as mean ± SD, n = 3. ( c ) Binding of TPPP/p25 (250 nM) and tubulin (1 μM) to the immobilized SIRT2 as detected by TPPP/p25 and tubulin antibodies, respectively. The data are presented as mean ± SD, n = 4. *p = 2.61E-04 when TPPP/p25 with tubulin compared to TPPP/p25 alone, and *p = 1.76E-02 when tubulin with TPPP/p25 compared to tubulin alone (two-sided, unpaired Student’s t-test).

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Interaction and localization of TPPP/p25 and SIRT2 in living HeLa cells as detected by immunofluorescence microscopy coupled with BiFC technology. ( a ) Scheme of the applied BiFC constructs. ( b ) Co-localization of the TPPP/p25-SIRT2 complex (green) with the MT network (red). MT network was stained with Alexa546, nuclei was counterstained with DAPI (blue). Scale bar: 10 μm. ( c ) BiFC signal (green) of the assembly of Venus N -TPPP/p25 and Venus C -SIRT2 and the effect of unlabelled TPPP/p25, α-synuclein and MZ25 was quantified as described in the Materials and Methods. The data are presented as mean ± SD, in each case at least 90 cells were analysed. *p = 2.45E-21 when control (BiFC) cells were compared with those co-transfected with unlabelled TPPP/p25 and *p = 6.76E-06 with unlabelled α-synuclein (two-sided, unpaired Student’s t-test).

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Interaction and localization of TPPP/p25 and SIRT2 in living HeLa cells as detected by immunofluorescence microscopy coupled with BiFC technology. ( a ) Scheme of the applied BiFC constructs. ( b ) Co-localization of the TPPP/p25-SIRT2 complex (green) with the MT network (red). MT network was stained with Alexa546, nuclei was counterstained with DAPI (blue). Scale bar: 10 μm. ( c ) BiFC signal (green) of the assembly of Venus N -TPPP/p25 and Venus C -SIRT2 and the effect of unlabelled TPPP/p25, α-synuclein and MZ25 was quantified as described in the Materials and Methods. The data are presented as mean ± SD, in each case at least 90 cells were analysed. *p = 2.45E-21 when control (BiFC) cells were compared with those co-transfected with unlabelled TPPP/p25 and *p = 6.76E-06 with unlabelled α-synuclein (two-sided, unpaired Student’s t-test).

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Immunofluorescence, Microscopy, Bimolecular Fluorescence Complementation Assay, Construct, Staining, Transfection

    Visualization of the acetylation of the MT network produced by SIRT2 inhibitors (MZ242 and SH1 PROTAC) in TPPP/p25 expressing (green) HeLa cells as compared to the non-transfected cells by widefield immunofluorescence microscopy using specific acetyl-tubulin antibody (red). ( a ) Cells treated with MZ242. ( c ) Cells treated with SH1. Note that the chemical compounds are taken up from the medium, while TPPP/p25 is expressed only in some of the cells. Nuclei are counterstained with DAPI (blue). Scale bar: 5 μm. The acetyl-tubulin signal (red) was quantified in the transfected and non-transfected cells as described in the Materials and Methods. The data are presented as mean ± SD, at least 25 cells (in the case of MZ242, b ) or 50 cells (in the case of SH1, d ) were analysed. *p = 9.20E-3 and *p = 3.34E-5 when TPPP/p25 transfected cells were compared with those treated with MZ242 and SH1 as well, respectively (two-sided, unpaired Student’s t-test).

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Visualization of the acetylation of the MT network produced by SIRT2 inhibitors (MZ242 and SH1 PROTAC) in TPPP/p25 expressing (green) HeLa cells as compared to the non-transfected cells by widefield immunofluorescence microscopy using specific acetyl-tubulin antibody (red). ( a ) Cells treated with MZ242. ( c ) Cells treated with SH1. Note that the chemical compounds are taken up from the medium, while TPPP/p25 is expressed only in some of the cells. Nuclei are counterstained with DAPI (blue). Scale bar: 5 μm. The acetyl-tubulin signal (red) was quantified in the transfected and non-transfected cells as described in the Materials and Methods. The data are presented as mean ± SD, at least 25 cells (in the case of MZ242, b ) or 50 cells (in the case of SH1, d ) were analysed. *p = 9.20E-3 and *p = 3.34E-5 when TPPP/p25 transfected cells were compared with those treated with MZ242 and SH1 as well, respectively (two-sided, unpaired Student’s t-test).

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Produced, Expressing, Transfection, Immunofluorescence, Microscopy

    Mathematical model of the binary and ternary complexes and comparison with the ELISA experiment. ( a ) Concentration of the different complexes according to the binary (dashed lines) and ternary (solid lines) models. ( b ) SIRT2 was immobilized on the plate, then tubulin without (○) or with 200 nM TPPP/p25 (●) was added and the bound tubulin was detected by tubulin antibody. The data are presented as mean ± SD, n = 4 for tubulin, n = 3 for tubulin with TPPP/p25. SIRT2-tubulin at 0 μM TPPP/p25: relative concentration of SIRT2-tubulin complex without added TPPP/p25. Relative concentrations of SIRT2-tubulin, TPPP/p25-SIRT2-tubulin and the sum of SIRT2-tubulin and TPPP/p25-SIRT2-tubulin according to the ternary model, respectively, are shown. Relative concentration of SIRT2-tubulin according to the binary model is also displayed.

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Mathematical model of the binary and ternary complexes and comparison with the ELISA experiment. ( a ) Concentration of the different complexes according to the binary (dashed lines) and ternary (solid lines) models. ( b ) SIRT2 was immobilized on the plate, then tubulin without (○) or with 200 nM TPPP/p25 (●) was added and the bound tubulin was detected by tubulin antibody. The data are presented as mean ± SD, n = 4 for tubulin, n = 3 for tubulin with TPPP/p25. SIRT2-tubulin at 0 μM TPPP/p25: relative concentration of SIRT2-tubulin complex without added TPPP/p25. Relative concentrations of SIRT2-tubulin, TPPP/p25-SIRT2-tubulin and the sum of SIRT2-tubulin and TPPP/p25-SIRT2-tubulin according to the ternary model, respectively, are shown. Relative concentration of SIRT2-tubulin according to the binary model is also displayed.

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

    Modest inhibitory potency of TPPP/p25 on the deacetylation of tubulin by SIRT2. ( a ) Concentration-dependent tubulin deacetylation by SIRT2 (●) and the inhibitory effect of TPPP/p25 (∆) on the deacetylase activity of 1.5 μM SIRT2 (indicated by an arrow). The acetylation was determined by Western blot using acetyl-tubulin antibody at 2.64 μM tubulin concentration. ( b ) Quantification of the Western blot data as described in the Materials and Methods. The data are presented as mean ± SD, n = 3 for the SIRT2 concentration serie, and n = 4 for the TPPP/p25 concentration serie at 1.5 μM SIRT2 concentration. Images of the full-length blots/gels are presented in Supplementary Fig. S4 .

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Modest inhibitory potency of TPPP/p25 on the deacetylation of tubulin by SIRT2. ( a ) Concentration-dependent tubulin deacetylation by SIRT2 (●) and the inhibitory effect of TPPP/p25 (∆) on the deacetylase activity of 1.5 μM SIRT2 (indicated by an arrow). The acetylation was determined by Western blot using acetyl-tubulin antibody at 2.64 μM tubulin concentration. ( b ) Quantification of the Western blot data as described in the Materials and Methods. The data are presented as mean ± SD, n = 3 for the SIRT2 concentration serie, and n = 4 for the TPPP/p25 concentration serie at 1.5 μM SIRT2 concentration. Images of the full-length blots/gels are presented in Supplementary Fig. S4 .

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Concentration Assay, Histone Deacetylase Assay, Activity Assay, Western Blot

    Secondary structural characteristics of SIRT2 and its complexes with tubulin and TPPP/p25 as detected by far-UV CD spectroscopy. ( a ) Normalized CD spectrum of SIRT2 (bold line), TPPP/p25 (solid line) and tubulin (dashed line) (n = 3); ( b ) Difference spectrum of SIRT2-tubulin (bold line), SIRT2-TPPP/p25 (solid line) and tubulin-TPPP/p25 (dashed line) (n = 3). Difference ellipticities were calculated by subtracting the ellipticities of the individual protein from that of their complexes. Concentrations of the proteins: 16 μM SIRT2, 4 μM TPPP/p25 and 1 μM tubulin.

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Secondary structural characteristics of SIRT2 and its complexes with tubulin and TPPP/p25 as detected by far-UV CD spectroscopy. ( a ) Normalized CD spectrum of SIRT2 (bold line), TPPP/p25 (solid line) and tubulin (dashed line) (n = 3); ( b ) Difference spectrum of SIRT2-tubulin (bold line), SIRT2-TPPP/p25 (solid line) and tubulin-TPPP/p25 (dashed line) (n = 3). Difference ellipticities were calculated by subtracting the ellipticities of the individual protein from that of their complexes. Concentrations of the proteins: 16 μM SIRT2, 4 μM TPPP/p25 and 1 μM tubulin.

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Spectroscopy

    Effect of TPPP/p25 and/or chemical inhibitors on the SIRT2 activity on tubulin. ( a ) Inhibition of deacetylation of 2.64 µM tubulin (black column) by 20 µM TPPP/p25 or chemical compounds; 0.6 µM SIRT2 was added to the acetyl-tubulin. The data are presented as mean ± SD, n = 3–11. *p = 5.56E-06 for TPPP/p25, *p = 1.53E-03 for SH1, *p = 1.38E-03 for MZ242 and *p = 1.60E-03 for 1 mM nicotinamide when the values with and without inhibitor were compared in the case of tubulin as a substrate (two-sided, unpaired Student’s t-test). Images of the full-length blots/gels are presented in Supplementary Fig. S5 . ( b ) Effect of the inhibitors on the binding of 125 nM TPPP/p25 to the immobilized SIRT2 detected by ELISA as described in the Materials and Methods. The data are presented as mean ± SD, n = 3. ( c ) Mutual effect of the TPPP/p25 and the chemical inhibitors on the SIRT2 activity (Western blot data with tubulin). The data are presented as mean ± SD. n = 4 for tubulin, tubulin with SIRT2, tubulin with SIRT2 and 10 μM TPPP/p25, tubulin with SIRT2 and 5 μM MZ242, and tubulin with SIRT2 and 10 μM TPPP/p25 and 5 μM MZ242; n = 3 for tubulin with SIRT2 and 5 μM SH1, and tubulin with SIRT2 and 10 μM TPPP/p25 and 5 μM SH1. *p = 1.06E-05 for TPPP/p25, *p = 4.96E-03 for MZ242 and *p = 1.38E-03 for SH1 when the values with and without inhibitor were compared (two-sided, unpaired Student’s t-test). Images of the full-length blots/gels are presented in Supplementary Fig. S6 .

    Journal: Scientific Reports

    Article Title: Modulation Of Microtubule Acetylation By The Interplay Of TPPP/p25, SIRT2 And New Anticancer Agents With Anti-SIRT2 Potency

    doi: 10.1038/s41598-017-17381-3

    Figure Lengend Snippet: Effect of TPPP/p25 and/or chemical inhibitors on the SIRT2 activity on tubulin. ( a ) Inhibition of deacetylation of 2.64 µM tubulin (black column) by 20 µM TPPP/p25 or chemical compounds; 0.6 µM SIRT2 was added to the acetyl-tubulin. The data are presented as mean ± SD, n = 3–11. *p = 5.56E-06 for TPPP/p25, *p = 1.53E-03 for SH1, *p = 1.38E-03 for MZ242 and *p = 1.60E-03 for 1 mM nicotinamide when the values with and without inhibitor were compared in the case of tubulin as a substrate (two-sided, unpaired Student’s t-test). Images of the full-length blots/gels are presented in Supplementary Fig. S5 . ( b ) Effect of the inhibitors on the binding of 125 nM TPPP/p25 to the immobilized SIRT2 detected by ELISA as described in the Materials and Methods. The data are presented as mean ± SD, n = 3. ( c ) Mutual effect of the TPPP/p25 and the chemical inhibitors on the SIRT2 activity (Western blot data with tubulin). The data are presented as mean ± SD. n = 4 for tubulin, tubulin with SIRT2, tubulin with SIRT2 and 10 μM TPPP/p25, tubulin with SIRT2 and 5 μM MZ242, and tubulin with SIRT2 and 10 μM TPPP/p25 and 5 μM MZ242; n = 3 for tubulin with SIRT2 and 5 μM SH1, and tubulin with SIRT2 and 10 μM TPPP/p25 and 5 μM SH1. *p = 1.06E-05 for TPPP/p25, *p = 4.96E-03 for MZ242 and *p = 1.38E-03 for SH1 when the values with and without inhibitor were compared (two-sided, unpaired Student’s t-test). Images of the full-length blots/gels are presented in Supplementary Fig. S6 .

    Article Snippet: Tubulin SIRT2 was incubated with 2.64 μM tubulin, 0.5 mM NAD+ , 0.1 mg/ml bovine serum albumin without or with TPPP/p25 and/or drugs in assay buffer (BPS Bioscience, 50031) for 1 h at 37 °C.

    Techniques: Activity Assay, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    Histogram of HMT G9a primary screening results

    Journal: Journal of biomolecular screening

    Article Title: Development and validation of a generic fluorescent methyltransferase activity assay based on the Transcreener® AMP/GMP Assay

    doi: 10.1177/1087057111421624

    Figure Lengend Snippet: Histogram of HMT G9a primary screening results

    Article Snippet: G9a HMT, SUV39H1 HMT, DNMT1 MT, and DNMT3a MT purchased from BPS Biosciences (San Diego, CA) and Set7/Set9 HMT was from Prospec Protein Specialists (Ness-Ziona, Israel).

    Techniques: HMT Assay

    Percent enzyme activity remaining for HDAC-1 (top, 0.25 μM) and HDAC-6 (bottom, 0.16 μM) in the presence of 53 or 22 nM SAHA (IC50 = 53 nM and 22 nM, respectively) and increasing concentrations of MT. The ratios of MPi to MT are indicated

    Journal: ChemMedChem

    Article Title: Investigating the Selectivity of Metalloenzyme Inhibitors in the Presence of Competing Metalloproteins

    doi: 10.1002/cmdc.201500293

    Figure Lengend Snippet: Percent enzyme activity remaining for HDAC-1 (top, 0.25 μM) and HDAC-6 (bottom, 0.16 μM) in the presence of 53 or 22 nM SAHA (IC50 = 53 nM and 22 nM, respectively) and increasing concentrations of MT. The ratios of MPi to MT are indicated

    Article Snippet: Each well contained a volume of 50 μl including buffer (BPS Bioscience, catalogue no. 50031), the mixture of proteins (HDAC-1 (0.033 mg/ml, BPS Bioscience, catalogue no. 50051) or HDAC-6 (0.025 mg/ml, BPS Bioscience, catalogue no. 50006) and competing proteins), inhibitor (at the IC50), BSA (1 mg/mL, Sigma-Aldrich), and HDAC substrate 3 (20 μM, BPS Bioscience, catalogue no. 50037).

    Techniques: Activity Assay