histone h3  (New England Biolabs)


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    New England Biolabs histone h3
    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone <t>H3</t> Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.
    Histone H3, 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
    histone h3 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability"

    Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.1018161

    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.
    Figure Legend Snippet: Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

    Techniques Used: Expressing, Activity Assay, Infection, Western Blot, Immunofluorescence

    Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.
    Figure Legend Snippet: Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.

    Techniques Used: Expressing, Activity Assay, Inhibition, Western Blot, Over Expression, Immunofluorescence

    histone h3  (New England Biolabs)


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    New England Biolabs histone h3
    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone <t>H3</t> Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.
    Histone H3, 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/histone h3/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability"

    Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.1018161

    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.
    Figure Legend Snippet: Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

    Techniques Used: Expressing, Activity Assay, Infection, Western Blot, Immunofluorescence

    Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.
    Figure Legend Snippet: Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.

    Techniques Used: Expressing, Activity Assay, Inhibition, Western Blot, Over Expression, Immunofluorescence

    m2506s  (New England Biolabs)


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    New England Biolabs m2506s
    KEY RESOURCES TABLE
    M2506s, 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/m2506s/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m2506s - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity"

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.110482

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    recombinant human h3 2  (New England Biolabs)


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    New England Biolabs recombinant human h3 2
    Recombinant Human H3 2, 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/recombinant human h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
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    recombinant histone h3 2  (New England Biolabs)


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    New England Biolabs recombinant histone h3 2
    Recombinant Histone H3 2, 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/recombinant histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
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    recombinant histone h3 2  (New England Biolabs)


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    New England Biolabs recombinant histone h3 2
    Recombinant Histone H3 2, 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/recombinant histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
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    human histone h3 2  (New England Biolabs)


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    New England Biolabs human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Human Histone H3 2, 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/human histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human histone h3 2 - by Bioz Stars, 2023-01
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    Images

    1) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal:

    doi: 10.1016/j.jnutbio.2010.04.001

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Incubation, Recombinant, Generated, Staining

    recombinant human histone h3 2  (New England Biolabs)


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    New England Biolabs recombinant human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Recombinant Human Histone H3 2, 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/recombinant human histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h3 2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal:

    doi: 10.1016/j.jnutbio.2010.04.001

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Incubation, Recombinant, Generated, Staining

    human histone h3 2  (New England Biolabs)


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  • 94

    Structured Review

    New England Biolabs human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Human Histone H3 2, 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/human histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human histone h3 2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal:

    doi: 10.1016/j.jnutbio.2010.04.001

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Incubation, Recombinant, Generated, Staining

    recombinant human histone h3 2  (New England Biolabs)


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    New England Biolabs recombinant human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Recombinant Human Histone H3 2, 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/recombinant human histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human histone h3 2 - by Bioz Stars, 2023-01
    94/100 stars

    Images

    1) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal:

    doi: 10.1016/j.jnutbio.2010.04.001

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Incubation, Recombinant, Generated, Staining

    histone h3 2  (New England Biolabs)


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    New England Biolabs histone h3 2
    Histone H3 2, 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/histone h3 2/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone h3 2 - by Bioz Stars, 2023-01
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    New England Biolabs histone h3
    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone <t>H3</t> Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.
    Histone H3, 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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    New England Biolabs m2506s
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    New England Biolabs recombinant human h3 2
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    Recombinant Human H3 2, 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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    KEY RESOURCES TABLE
    Recombinant Histone H3 2, 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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    New England Biolabs human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Human Histone H3 2, 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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    New England Biolabs recombinant human histone h3 2
    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Recombinant Human Histone H3 2, 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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    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone <t>H3.2</t> (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Histone H3 2, 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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    Image Search Results


    Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

    doi: 10.3389/fcell.2022.1018161

    Figure Lengend Snippet: Expression of GFP-Aurora B in HeLa cells causes a reduction in Aurora B kinase activity and an increase in mitotic defects. Cells were analyzed 48 h after infection with retroviruses expressing GFP-Aurora B or GFP alone. (A) Immunoblot showing endogenous pT232 (active) Aurora B is reduced in HeLa cells overexpressing Aurora B. Tubulin is shown as a loading control. (B) Immunoblot showing reduced phosphorylation of the bona fide Aurora B substrate histone H3 Serine 10 in HeLa cells infected with retroviruses expressing GFP-Aurora. Tubulin is shown as a loading control. (C–H) Quantitative immunofluorescence demonstrating that expression of GFP-Aurora B decreases phosphorylation of histone H3 (pH3), the kinetochore component DSN1, and INCENP in HeLa cells. (C,E,G) Representative images of pH3, pDSN1, and pINCENP, respectively, in HeLa cells expressing GFP and GFP-Aurora B. (D,F,H) Quantification of pH3 S10 (D) , pDSN1 S109 (F) , and pINCENP TSS (H) demonstrating that expression of GFP-Aurora B significantly reduces phosphorylation of these Aurora B substrates in HeLa cells. n > 10 cells from each of 3 independent experiments. (I–P) GFP-Aurora B expression increases the incidence of mitotic defects. (I) Representative image of a misaligned chromosome (arrow). (J) Expression of GFP-Aurora B in HeLa cells causes an increase in misaligned chromosomes in metaphase. n > 50 metaphase cells from 3 independent experiments. (K) Representative image of a lagging chromosome (arrow). (L) Expression GFP-Aurora B in HeLa cells causes an increase in lagging chromosomes. n > 250 anaphase and telophase cells from each of 3 independent experiments. (M) Representative image of a multipolar spindle. (N) Expression of GFP-Aurora B in HeLa cells causes an increase in multipolar spindles. n > 250 mitotic cells from each of 3 independent experiments. (O) Expression of GFP-Aurora B increases the percentage of binucleate HeLa cells. n > 250 cells from each of three independent experiments. (P) Expression of GFP-Aurora B in HeLa cells impairs their ability to arrest in mitosis after 16 h of treatment with the microtubule poison colcemid, consistent with a weakened mitotic checkpoint. n > 250 cells from each of 3 independent experiments. * p < 0.05.

    Article Snippet: Aurora B-3xFLAG was immunoprecipitated with M2-agarose slurry (Selleck B23102) and incubated in buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 1.5M NaCl, 100 mM NaF) with 1 mM DTT, 1 μM cold ATP, 5 μCi [γ- 32 P] ATP, 2.5 μg histone H3 (NEB M2506S) and with or without the IN-box of recombinant INCENP (Signal Chem I30-31H) at 30°C for 30 min and resolved by SDS-PAGE. γ- 32 P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).

    Techniques: Expressing, Activity Assay, Infection, Western Blot, Immunofluorescence

    Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Increased Aurora B expression reduces substrate phosphorylation and induces chromosomal instability

    doi: 10.3389/fcell.2022.1018161

    Figure Lengend Snippet: Increased expression of untagged Aurora B inhibits Aurora B kinase activity and causes resistance to Aurora kinase inhibition. (A) Immunoblot showing ≥2- fold overexpression of untagged Aurora B in MDA-MB-231 cells treated with tetracycline. Despite increased protein levels of Aurora B, pT232 (active) Aurora B is reduced. Tubulin is shown as a loading control. (B–E) Quantitative immunofluorescence showing that increased Aurora B expression results in reduced phosphorylation of the Aurora B substrates histone H3 and KNL1. (B,D) Representative images of pH3 and pKNL1 in MDA-MB-231 cells ± 48 h of tet-inducible expression of Aurora B (C,E) Quantification of B and D. n > 10 cells from each of 3 independent experiments. (F–I) 48 h of tet-induced Aurora B expression induces mitotic defects in MDA-MB-231 cells. (F) Representative image of a misaligned chromosome (arrow). (G) Elevated Aurora B expression increases the incidence of misaligned chromosomes. n > 50 metaphases from 3 independent experiments. (H) Representative image of a lagging chromosome (arrow). (I) Increased Aurora B expression induces lagging chromosomes. n > 100 anaphase and telophases from 3 independent experiments. (J–O) Increased Aurora B expression causes resistance to Aurora B inhibition. (J) Primary ARF −/− MEFs grow significantly better than ARF +/+ MEFS after 16 h of exposure to the Aurora B inhibitor ZM447439. Cell number shown is normalized to the number of ARF +/+ cells 10 days after treatment with DMSO. n = 3 independent experiments. (K) Primary ARF −/− MEFs (red), which overexpress endogenous Aurora B, have significantly improved colony forming ability as compared to ARF +/+ MEFs (blue) after 16 h exposure to ZM447439. n = 3 independent experiments. (L) MDA-MB-231 cells expressing untagged Aurora B in response to tetracycline grow significantly better over the course of 10 days than controls after 16 h exposure to ZM447439. n = 3 independent experiments. (M) MDA-MB-231 cells expressing tet-inducible wild type Aurora B exhibit significantly elevated colony forming ability compared to control MDA-MB-231 cells after exposure to ZM447439. n = 3 independent experiments. (N) MCF7 cells expressing Aurora B–GFP in response to tetracycline grow significantly better over the course of 10 days than MCF7 controls after treatment with ZM447439. n = 3 independent experiments. (O) MCF7 cells expressing Aurora B–GFP form significantly more colonies than control cells after exposure to ZM447439. n = 3 independent experiments. * p < 0.05.

    Article Snippet: Aurora B-3xFLAG was immunoprecipitated with M2-agarose slurry (Selleck B23102) and incubated in buffer (200 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 1.5M NaCl, 100 mM NaF) with 1 mM DTT, 1 μM cold ATP, 5 μCi [γ- 32 P] ATP, 2.5 μg histone H3 (NEB M2506S) and with or without the IN-box of recombinant INCENP (Signal Chem I30-31H) at 30°C for 30 min and resolved by SDS-PAGE. γ- 32 P incorporation was visualized by Typhoon TRIO imager (GE Healthcare).

    Techniques: Expressing, Activity Assay, Inhibition, Western Blot, Over Expression, Immunofluorescence

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

    doi: 10.1016/j.celrep.2022.110482

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: H3 (histone) , New England BioLabs , Cat# M2506S.

    Techniques: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Journal:

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Article Snippet: Ten micrograms of recombinant human histone H3.2 (New England Biolabs, Ipswich, MA) or 20 μg of peptide N 1–25 , which comprises the 25 N-terminal amino acids in H3 [ 9 ], were substituted for p67; the amount of rHCS in reaction mixtures was increased to 0.9 μg, and incubations were conducted for 12 h unless noted otherwise.

    Techniques: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Journal:

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Article Snippet: Ten micrograms of recombinant human histone H3.2 (New England Biolabs, Ipswich, MA) or 20 μg of peptide N 1–25 , which comprises the 25 N-terminal amino acids in H3 [ 9 ], were substituted for p67; the amount of rHCS in reaction mixtures was increased to 0.9 μg, and incubations were conducted for 12 h unless noted otherwise.

    Techniques: Incubation, Recombinant, Generated, Staining

    Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Journal:

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Article Snippet: Briefly, 3 μg recombinant human histone H3.2 (New England Biolabs) was incubated with or without 15 μg rHCS in 50 μl of 75 mM Tris acetate buffer (pH 7.5), containing 0.3 mM DTT, and 45 mM MgCl 2 , at 37°C for 2 h. Note that the concentrations of H3.2 and HCS are approximately equimolar in this sample.

    Techniques: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Journal:

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    doi: 10.1016/j.jnutbio.2010.04.001

    Figure Lengend Snippet: Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N1–25) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Article Snippet: Briefly, 3 μg recombinant human histone H3.2 (New England Biolabs) was incubated with or without 15 μg rHCS in 50 μl of 75 mM Tris acetate buffer (pH 7.5), containing 0.3 mM DTT, and 45 mM MgCl 2 , at 37°C for 2 h. Note that the concentrations of H3.2 and HCS are approximately equimolar in this sample.

    Techniques: Incubation, Recombinant, Generated, Staining