histone h2a  (Cell Signaling Technology Inc)

 
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    Name:
    Histone H2A Antibody II
    Description:
    Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes The nucleosome made up of DNA wound around eight core histone proteins two each of H2A H2B H3 and H4 is the primary building block of chromatin 1 The amino terminal tails of core histones undergo various post translational modifications including acetylation phosphorylation methylation and ubiquitination 2 5 These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and therefore gene expression 6 In most species histone H2B is primarily acetylated at Lys5 12 15 and 20 4 7 Histone H3 is primarily acetylated at Lys9 14 18 23 27 and 56 Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms 2 3 Phosphorylation at Ser10 Ser28 and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis 8 10 Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin Immunostaining with phospho specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase 11
    Catalog Number:
    2578
    Price:
    None
    Applications:
    Western Blot, Immunoprecipitation, Immunohistochemistry
    Category:
    Primary Antibodies
    Source:
    Polyclonal antibodies are produced by immunizing animals with a synthetic peptide designed based on the region around Threonine 77 of human Histone H2A sequence. Antibodies are purified by protein A and peptide affinity chromatography.
    Reactivity:
    Human Mouse Rat Monkey Zebrafish
    Buy from Supplier


    Structured Review

    Cell Signaling Technology Inc histone h2a
    Analysis of adsorbed proteins identified with mass spectrometry. Proteins adsorbed on the surface of each multi‐wall carbon nanotubes (MWCNT) were identified with liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). The results from each sample were compared for overlap (a). Three histones <t>(H2A,</t> H2B and H3), two subunits of hemoglobin (Hb‐α and Hb‐β) and three other proteins (Tf, transferrin; Prdx6, peroxiredoxin 6; Keap1, Kelch‐like ECH‐associated protein 1) associated with oxidative stress and based on our previous experiments on asbestos were picked from the common cluster and were confirmed with western blotting analysis (b). Please refer to the text for details.
    Modulation of chromatin structure plays an important role in the regulation of transcription in eukaryotes The nucleosome made up of DNA wound around eight core histone proteins two each of H2A H2B H3 and H4 is the primary building block of chromatin 1 The amino terminal tails of core histones undergo various post translational modifications including acetylation phosphorylation methylation and ubiquitination 2 5 These modifications occur in response to various stimuli and have a direct effect on the accessibility of chromatin to transcription factors and therefore gene expression 6 In most species histone H2B is primarily acetylated at Lys5 12 15 and 20 4 7 Histone H3 is primarily acetylated at Lys9 14 18 23 27 and 56 Acetylation of H3 at Lys9 appears to have a dominant role in histone deposition and chromatin assembly in some organisms 2 3 Phosphorylation at Ser10 Ser28 and Thr11 of histone H3 is tightly correlated with chromosome condensation during both mitosis and meiosis 8 10 Phosphorylation at Thr3 of histone H3 is highly conserved among many species and is catalyzed by the kinase haspin Immunostaining with phospho specific antibodies in mammalian cells reveals mitotic phosphorylation at Thr3 of H3 in prophase and its dephosphorylation during anaphase 11
    https://www.bioz.com/result/histone h2a/product/Cell Signaling Technology Inc
    Average 99 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    histone h2a - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Role of hemoglobin and transferrin in multi‐wall carbon nanotube‐induced mesothelial injury and carcinogenesis"

    Article Title: Role of hemoglobin and transferrin in multi‐wall carbon nanotube‐induced mesothelial injury and carcinogenesis

    Journal: Cancer Science

    doi: 10.1111/cas.12865

    Analysis of adsorbed proteins identified with mass spectrometry. Proteins adsorbed on the surface of each multi‐wall carbon nanotubes (MWCNT) were identified with liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). The results from each sample were compared for overlap (a). Three histones (H2A, H2B and H3), two subunits of hemoglobin (Hb‐α and Hb‐β) and three other proteins (Tf, transferrin; Prdx6, peroxiredoxin 6; Keap1, Kelch‐like ECH‐associated protein 1) associated with oxidative stress and based on our previous experiments on asbestos were picked from the common cluster and were confirmed with western blotting analysis (b). Please refer to the text for details.
    Figure Legend Snippet: Analysis of adsorbed proteins identified with mass spectrometry. Proteins adsorbed on the surface of each multi‐wall carbon nanotubes (MWCNT) were identified with liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). The results from each sample were compared for overlap (a). Three histones (H2A, H2B and H3), two subunits of hemoglobin (Hb‐α and Hb‐β) and three other proteins (Tf, transferrin; Prdx6, peroxiredoxin 6; Keap1, Kelch‐like ECH‐associated protein 1) associated with oxidative stress and based on our previous experiments on asbestos were picked from the common cluster and were confirmed with western blotting analysis (b). Please refer to the text for details.

    Techniques Used: Mass Spectrometry, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Western Blot

    2) Product Images from "Targeting STAT3/miR-21 axis inhibits epithelial-mesenchymal transition via regulating CDK5 in head and neck squamous cell carcinoma"

    Article Title: Targeting STAT3/miR-21 axis inhibits epithelial-mesenchymal transition via regulating CDK5 in head and neck squamous cell carcinoma

    Journal: Molecular Cancer

    doi: 10.1186/s12943-015-0487-x

    miR-21 blockade reverses EMT by regulating CDK5/p35 in HNSCC cells in vitro. a Expression of EMT-related proteins after asON or WP1066 treatment, analyzed by western blot with normalization to β-actin. b Expressions of CDK5 and p35 after asON and WP1066 treatment, determined by western blot with normalization to β-actin. c β-catenin level in nucleus and cytoplasm, analyzed by western blot with normalization to Histone H2A. d Subcellular location of EMT related markers after asON or WP1066 treatment, observed by immunofluorescence staining (original magnification, x100)
    Figure Legend Snippet: miR-21 blockade reverses EMT by regulating CDK5/p35 in HNSCC cells in vitro. a Expression of EMT-related proteins after asON or WP1066 treatment, analyzed by western blot with normalization to β-actin. b Expressions of CDK5 and p35 after asON and WP1066 treatment, determined by western blot with normalization to β-actin. c β-catenin level in nucleus and cytoplasm, analyzed by western blot with normalization to Histone H2A. d Subcellular location of EMT related markers after asON or WP1066 treatment, observed by immunofluorescence staining (original magnification, x100)

    Techniques Used: In Vitro, Expressing, Western Blot, Immunofluorescence, Staining

    3) Product Images from "Acetylome Analysis Identifies SIRT1 Targets in mRNA-Processing and Chromatin-Remodeling in Mouse Liver"

    Article Title: Acetylome Analysis Identifies SIRT1 Targets in mRNA-Processing and Chromatin-Remodeling in Mouse Liver

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0140619

    SIRT1 deacetylates hnRNP L and hnRNP C1/C2 in vivo . Acetylated proteins were immunoprecipitated using the pan-acetyl-lysine antibody from the liver extracts of SIRT1 +/+ and Sirt1 -/- mice. The amounts of acetylated hnRNP L and hnRNP C1/C2 proteins were evaluated by their corresponding antibodies in the IP samples. The protein levels of hnRNP L, hnRNP C1/C2 and SIRT1 were assessed by western blotting using the corresponding antibodies. Histone H2A was used as a loading control.
    Figure Legend Snippet: SIRT1 deacetylates hnRNP L and hnRNP C1/C2 in vivo . Acetylated proteins were immunoprecipitated using the pan-acetyl-lysine antibody from the liver extracts of SIRT1 +/+ and Sirt1 -/- mice. The amounts of acetylated hnRNP L and hnRNP C1/C2 proteins were evaluated by their corresponding antibodies in the IP samples. The protein levels of hnRNP L, hnRNP C1/C2 and SIRT1 were assessed by western blotting using the corresponding antibodies. Histone H2A was used as a loading control.

    Techniques Used: In Vivo, Immunoprecipitation, Mouse Assay, Western Blot

    4) Product Images from "Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells"

    Article Title: Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2014.08.037

    Co-localization of fetuin-A and histone H2A in the cell nucleus
    Figure Legend Snippet: Co-localization of fetuin-A and histone H2A in the cell nucleus

    Techniques Used:

    Localization of fetuin-A and histone H2A on the extracellular surfaces of cellular exosomes by FAVS
    Figure Legend Snippet: Localization of fetuin-A and histone H2A on the extracellular surfaces of cellular exosomes by FAVS

    Techniques Used:

    Fetuin-A mediated trafficking of vinculin and histone H2A to the membrane fraction of adhered and spread cells
    Figure Legend Snippet: Fetuin-A mediated trafficking of vinculin and histone H2A to the membrane fraction of adhered and spread cells

    Techniques Used:

    5) Product Images from "Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling"

    Article Title: Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.21332

    Actions of DCT on phosphorylation of GSK and BAD, and on nuclear translocation of NF-κB. A, a : HT-29 and H508 cells were incubated with DCT(300 µM) at 37°C for up to 16 h and cell extracts were immunoblotted at the times indicated to determine changes in phospho-GSK Ser 21/9 (p-GSK). The quantity of protein added was verified by immunoblotting with antibodies for total GSK. b : Control HT-29 cells and HT-29 cells transfected with plasmids containing wild-type (WT) and mutant (DNM) AKT ) were incubated with DCT for 2 h at 37°C and cell extracts were immunoblotted to determine changes in phospho-GSK. The quantity of protein added was verified by immunoblotting with antibody for total GSK. c : Graphic representation of densitometry data from immunoblots prepared as described in b . Densitometry units are arbitrary based on the value for the control lane (untransfected HT-29 cells treated with water) defined as I. Results are expressed as mean ± SD of at least three separate experiments. B: HT-29 cells were incubated with 300 µM DCT for up to 16 h at 37°C and cell extracts were immunoblotted at the times indicated to determine changes in phospho-BAD Ser 136 (p-BAD). The quantity of protein added was verified by immunoblotting with antibody for total BAD. C: HT-29 and H508 cells were pre-incubated with the indicated concentrations of PI3K inhibitors for 30 min and then incubated with or without DCT (100 µM) for 2 h at 37°C. Nuclear extracts were immunoblotted for NF-κB as described in Methods Section. The quantity of protein added was verified by immunoblotting using antibody for a nuclear protein, histone H2A. Results shown are representative of three separate experiments.
    Figure Legend Snippet: Actions of DCT on phosphorylation of GSK and BAD, and on nuclear translocation of NF-κB. A, a : HT-29 and H508 cells were incubated with DCT(300 µM) at 37°C for up to 16 h and cell extracts were immunoblotted at the times indicated to determine changes in phospho-GSK Ser 21/9 (p-GSK). The quantity of protein added was verified by immunoblotting with antibodies for total GSK. b : Control HT-29 cells and HT-29 cells transfected with plasmids containing wild-type (WT) and mutant (DNM) AKT ) were incubated with DCT for 2 h at 37°C and cell extracts were immunoblotted to determine changes in phospho-GSK. The quantity of protein added was verified by immunoblotting with antibody for total GSK. c : Graphic representation of densitometry data from immunoblots prepared as described in b . Densitometry units are arbitrary based on the value for the control lane (untransfected HT-29 cells treated with water) defined as I. Results are expressed as mean ± SD of at least three separate experiments. B: HT-29 cells were incubated with 300 µM DCT for up to 16 h at 37°C and cell extracts were immunoblotted at the times indicated to determine changes in phospho-BAD Ser 136 (p-BAD). The quantity of protein added was verified by immunoblotting with antibody for total BAD. C: HT-29 and H508 cells were pre-incubated with the indicated concentrations of PI3K inhibitors for 30 min and then incubated with or without DCT (100 µM) for 2 h at 37°C. Nuclear extracts were immunoblotted for NF-κB as described in Methods Section. The quantity of protein added was verified by immunoblotting using antibody for a nuclear protein, histone H2A. Results shown are representative of three separate experiments.

    Techniques Used: Translocation Assay, Incubation, Transfection, Mutagenesis, Western Blot

    6) Product Images from "Mitogen- and stress-activated Kinase 1 mediates Epstein-Barr virus latent membrane protein 1-promoted cell transformation in nasopharyngeal carcinoma through its induction of Fra-1 and c-Jun genes"

    Article Title: Mitogen- and stress-activated Kinase 1 mediates Epstein-Barr virus latent membrane protein 1-promoted cell transformation in nasopharyngeal carcinoma through its induction of Fra-1 and c-Jun genes

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1398-3

    Phosphorylation of histone H3 at Ser10 is involved in LMP1 induction of Fra-1 and c-Jun in CNE1 cells. ( a ) The expressions of H3 WT and H3 S10A mutant in stably transfected CNE1 cells were detected with an anti-His antibody against His-histone H3 by Western blotting. Histone H2A was used as loading control. ( b ) CNE1 cells stably expressing mock, H3 WT or mutant H3 S10A were transfected with pcDNA3.0 or pcDNA3.0-LMP1. At 36 hours after transfection, the firefly luciferase activity was measured and normalized against Renilla luciferase activity respectively. * P
    Figure Legend Snippet: Phosphorylation of histone H3 at Ser10 is involved in LMP1 induction of Fra-1 and c-Jun in CNE1 cells. ( a ) The expressions of H3 WT and H3 S10A mutant in stably transfected CNE1 cells were detected with an anti-His antibody against His-histone H3 by Western blotting. Histone H2A was used as loading control. ( b ) CNE1 cells stably expressing mock, H3 WT or mutant H3 S10A were transfected with pcDNA3.0 or pcDNA3.0-LMP1. At 36 hours after transfection, the firefly luciferase activity was measured and normalized against Renilla luciferase activity respectively. * P

    Techniques Used: Mutagenesis, Stable Transfection, Transfection, Western Blot, Expressing, Luciferase, Activity Assay

    7) Product Images from "Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)"

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003440

    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Figure Legend Snippet: Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.

    Techniques Used: Mutagenesis, Mass Spectrometry, Plasmid Preparation, Inhibition, Lysis, Affinity Purification, Western Blot, Staining, Immunolabeling, Fractionation, Marker

    8) Product Images from "Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis"

    Article Title: Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151136

    Analysis of V617F;Ezh2 double mutant mice. (A) Time course of blood counts and survival of MxCre mice induced with pIpC (yellow arrows). The color code for the different genotypes is the same as indicated in C–F. The gray horizontal bars indicate the normal range for the blood values. Survival probability was calculated by the Kaplan-Meier method. The means of the values ± SEM are plotted ( n = 4 for MxCre;GFP , n = 6 for MxCre;Ezh2 +/Δ ;GFP , n = 3 for MxCre;Ezh2 Δ/Δ ;GFP , n = 3 for MxCre;V617F;GFP , n = 4 for MxCre;V617F;Ezh2 +/Δ ;GFP , and n = 5 for MxCre;V617F;Ezh2 Δ/Δ ;GFP ). (B) Analysis of V617F;Ezh2 double mutant mice using the inducible SclCre system. Time course of blood counts of mice induced with tamoxifen injections for 5 wk with 1-wk break (orange horizontal bars). The means of the values ± SEM are plotted ( n = 6 for SclCre , n = 13 for SclCre;Ezh2 +/Δ , n = 8 for SclCre;Ezh2 Δ/Δ , n = 9 for SclCre;V617F , n = 12 for SclCre;V617F;Ezh2 +/Δ , and n = 14 for SclCre;V617F;Ezh2 Δ/Δ ). The same color code for the different genotypes is used. (C) A subset of mice was sacrificed at 12 wk after tamoxifen induction. Expression of Ezh2 mRNA was determined by real-time PCR in total BM and is shown in arbitrary units with WT (wt) control BM set to the value of 1. The group sizes were n = 5 for SclCre , n = 5 for SclCre;Ezh2 +/Δ , n = 4 for SclCre ; Ezh2 Δ/Δ , n = 4 for SclCre;V617F , n = 5 for SclCre;V617F;Ezh2 +/Δ , and n = 5 for SclCre;V617F;Ezh2 Δ/Δ . (D) Western blots of total BM cell lysates probed with antibodies against Ezh2, β-actin, H3K27me3, total histone H3, H2AK119Ub, and total H2A are shown. Genotypes are indicated above the corresponding lanes ( n = 3 for each group). This experiment was performed twice. (E and F) Spleen weight (E) and BM cellularity (F) are shown at 12 wk after tamoxifen ( n = 9 or 12 for SclCre , n = 7 or 12 for SclCre;Ezh2 +/Δ , n = 10 or 13 for SclCre;Ezh2 Δ/Δ , n = 5 or 8 for SclCre;V617F , n = 6 or 10 for SclCre;V617F;Ezh2 +/Δ , and n = 6 or 8 for SclCre;V617F;Ezh2 Δ/Δ for spleen weight or BM cellularity). This experiment was performed four times. (C, E, and F) Results are presented as means ± SEM. (G and H) Histopathology of BM and spleen taken at 16 wk after start of tamoxifen injection. The sections were stained with hematoxylin and eosin (H E) or with the Gömöri stain (Göm). The grade of MF is indicated under the corresponding pictures. Grading of MF was performed on three mice per group, and one representative mouse per genotype is shown. Bars: (black) 500 µm; (yellow) 50 µm; (blue) 20 µm; (orange) 10 µm. Statistical analysis was conducted using the Student’s t test or one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. *, P
    Figure Legend Snippet: Analysis of V617F;Ezh2 double mutant mice. (A) Time course of blood counts and survival of MxCre mice induced with pIpC (yellow arrows). The color code for the different genotypes is the same as indicated in C–F. The gray horizontal bars indicate the normal range for the blood values. Survival probability was calculated by the Kaplan-Meier method. The means of the values ± SEM are plotted ( n = 4 for MxCre;GFP , n = 6 for MxCre;Ezh2 +/Δ ;GFP , n = 3 for MxCre;Ezh2 Δ/Δ ;GFP , n = 3 for MxCre;V617F;GFP , n = 4 for MxCre;V617F;Ezh2 +/Δ ;GFP , and n = 5 for MxCre;V617F;Ezh2 Δ/Δ ;GFP ). (B) Analysis of V617F;Ezh2 double mutant mice using the inducible SclCre system. Time course of blood counts of mice induced with tamoxifen injections for 5 wk with 1-wk break (orange horizontal bars). The means of the values ± SEM are plotted ( n = 6 for SclCre , n = 13 for SclCre;Ezh2 +/Δ , n = 8 for SclCre;Ezh2 Δ/Δ , n = 9 for SclCre;V617F , n = 12 for SclCre;V617F;Ezh2 +/Δ , and n = 14 for SclCre;V617F;Ezh2 Δ/Δ ). The same color code for the different genotypes is used. (C) A subset of mice was sacrificed at 12 wk after tamoxifen induction. Expression of Ezh2 mRNA was determined by real-time PCR in total BM and is shown in arbitrary units with WT (wt) control BM set to the value of 1. The group sizes were n = 5 for SclCre , n = 5 for SclCre;Ezh2 +/Δ , n = 4 for SclCre ; Ezh2 Δ/Δ , n = 4 for SclCre;V617F , n = 5 for SclCre;V617F;Ezh2 +/Δ , and n = 5 for SclCre;V617F;Ezh2 Δ/Δ . (D) Western blots of total BM cell lysates probed with antibodies against Ezh2, β-actin, H3K27me3, total histone H3, H2AK119Ub, and total H2A are shown. Genotypes are indicated above the corresponding lanes ( n = 3 for each group). This experiment was performed twice. (E and F) Spleen weight (E) and BM cellularity (F) are shown at 12 wk after tamoxifen ( n = 9 or 12 for SclCre , n = 7 or 12 for SclCre;Ezh2 +/Δ , n = 10 or 13 for SclCre;Ezh2 Δ/Δ , n = 5 or 8 for SclCre;V617F , n = 6 or 10 for SclCre;V617F;Ezh2 +/Δ , and n = 6 or 8 for SclCre;V617F;Ezh2 Δ/Δ for spleen weight or BM cellularity). This experiment was performed four times. (C, E, and F) Results are presented as means ± SEM. (G and H) Histopathology of BM and spleen taken at 16 wk after start of tamoxifen injection. The sections were stained with hematoxylin and eosin (H E) or with the Gömöri stain (Göm). The grade of MF is indicated under the corresponding pictures. Grading of MF was performed on three mice per group, and one representative mouse per genotype is shown. Bars: (black) 500 µm; (yellow) 50 µm; (blue) 20 µm; (orange) 10 µm. Statistical analysis was conducted using the Student’s t test or one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. *, P

    Techniques Used: Mutagenesis, Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Histopathology, Injection, Staining

    9) Product Images from "The anti-cancer effect of probiotic Bacillus polyfermenticus on human colon cancer cells is mediated through ErbB2 and ErbB3 inhibition"

    Article Title: The anti-cancer effect of probiotic Bacillus polyfermenticus on human colon cancer cells is mediated through ErbB2 and ErbB3 inhibition

    Journal: International journal of cancer. Journal international du cancer

    doi: 10.1002/ijc.25011

    B.P. CM decreased E2F-1 and Cyclin D1 expression. (A) The HT-29 colon cancer cells were incubated with B.P. CM for 6 h, 18 h, 24 h, or 2 weeks. Nuclear extracts were made and subjected to the immumoblot analysis for E2F-1, β-actin or H2A. (B)
    Figure Legend Snippet: B.P. CM decreased E2F-1 and Cyclin D1 expression. (A) The HT-29 colon cancer cells were incubated with B.P. CM for 6 h, 18 h, 24 h, or 2 weeks. Nuclear extracts were made and subjected to the immumoblot analysis for E2F-1, β-actin or H2A. (B)

    Techniques Used: Expressing, Incubation

    10) Product Images from "Microinjection of Antibodies Targeting the Lamin A/C Histone-Binding Site Blocks Mitotic Entry and Reveals Separate Chromatin Interactions with HP1, CenpB and PML"

    Article Title: Microinjection of Antibodies Targeting the Lamin A/C Histone-Binding Site Blocks Mitotic Entry and Reveals Separate Chromatin Interactions with HP1, CenpB and PML

    Journal: Cells

    doi: 10.3390/cells6020009

    Lamin A/C histone binding site antibodies are specific, target in vivo and block H2A binding in vitro. ( a ) Schematic of lamin A domain structure highlighting the head, rod and tail domains, as well as the mapped chromatin binding site (HBS; Taniura et al., 1995). ( b ) Western blot on total HeLa cell lysates using a pan-lamin antibody and each histone-binding site antibody (A/C, B1 and B2) revealed that all antibodies prepared against the chromatin binding sites of lamins were each highly specific for each subtype. ( c ) The affinity-purified antibodies were conjugated to an SV40 NLS peptide and microinjected into either the nuclei or cytoplasm of U2OS cells. In both cases, subsequent fixation and visualization with fluorophore-conjugated secondary antibodies revealed nuclear rim staining, consistent with their binding the expected target on lamin A in the polymer underlying the inner nuclear membrane. ( d ) To test for antibody blocking of histone binding, lamin A∆rod protein was coupled to beads, incubated with either no antibodies (Lane 2), antibodies generated against BSA (Lane 3) or the lamin A histone-binding site antibodies (Lane 4). Uncoupled beads were also tested as a control for background binding to the beads because histones tend to be sticky (Lane 1). After washing, each was incubated with histones, eluted with SDS and analysed for the amount of bound histones by Western blot. Three technical replicates were quantified for fluorescence intensity using a LiCor Odyssey and plotted (n = 3). Standard deviations are shown, paired t-test shows a significant reduction in H2A bound by lamin A∆rod in the presence of the histone-binding site antibody compared to the BSA control. Scale bars, 10 µm.
    Figure Legend Snippet: Lamin A/C histone binding site antibodies are specific, target in vivo and block H2A binding in vitro. ( a ) Schematic of lamin A domain structure highlighting the head, rod and tail domains, as well as the mapped chromatin binding site (HBS; Taniura et al., 1995). ( b ) Western blot on total HeLa cell lysates using a pan-lamin antibody and each histone-binding site antibody (A/C, B1 and B2) revealed that all antibodies prepared against the chromatin binding sites of lamins were each highly specific for each subtype. ( c ) The affinity-purified antibodies were conjugated to an SV40 NLS peptide and microinjected into either the nuclei or cytoplasm of U2OS cells. In both cases, subsequent fixation and visualization with fluorophore-conjugated secondary antibodies revealed nuclear rim staining, consistent with their binding the expected target on lamin A in the polymer underlying the inner nuclear membrane. ( d ) To test for antibody blocking of histone binding, lamin A∆rod protein was coupled to beads, incubated with either no antibodies (Lane 2), antibodies generated against BSA (Lane 3) or the lamin A histone-binding site antibodies (Lane 4). Uncoupled beads were also tested as a control for background binding to the beads because histones tend to be sticky (Lane 1). After washing, each was incubated with histones, eluted with SDS and analysed for the amount of bound histones by Western blot. Three technical replicates were quantified for fluorescence intensity using a LiCor Odyssey and plotted (n = 3). Standard deviations are shown, paired t-test shows a significant reduction in H2A bound by lamin A∆rod in the presence of the histone-binding site antibody compared to the BSA control. Scale bars, 10 µm.

    Techniques Used: Binding Assay, In Vivo, Blocking Assay, In Vitro, Western Blot, Affinity Purification, Staining, Incubation, Generated, Fluorescence

    11) Product Images from "BMI-1 is a potential therapeutic target in diffuse intrinsic pontine glioma"

    Article Title: BMI-1 is a potential therapeutic target in diffuse intrinsic pontine glioma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18002

    PTC-209 reduces BMI-1 levels, PRC1 activity, and inhibits cell growth of DIPG neurospheres (A) Immunoblot analysis of BMI-1 and H2AK119Ub levels following 72 h treatment with 10 μM PTC-209. β-actin and total H2A served as loading controls. (B) Cell proliferation of DIPG cell lines treated for 72 hours with various concentrations of PTC-209 (DMSO, 1, 2, 5, 10 μM), measured using WST-1 assay and represented as percent (%) cell growth relative to DMSO treatment (control). (C–H) Representative images and quantification of neurosphere size (μm) and number of DIPG neurospheres treated with the indicated concentrations of PTC-209. CCHMC-DIPG-1 (C-D), CCHMC-DIPG-2 (E-F), and SU-DIPG-XXI (G-H). Error bars represent the standard deviation obtained from triplicates. Each experiment was performed at least twice. *, P
    Figure Legend Snippet: PTC-209 reduces BMI-1 levels, PRC1 activity, and inhibits cell growth of DIPG neurospheres (A) Immunoblot analysis of BMI-1 and H2AK119Ub levels following 72 h treatment with 10 μM PTC-209. β-actin and total H2A served as loading controls. (B) Cell proliferation of DIPG cell lines treated for 72 hours with various concentrations of PTC-209 (DMSO, 1, 2, 5, 10 μM), measured using WST-1 assay and represented as percent (%) cell growth relative to DMSO treatment (control). (C–H) Representative images and quantification of neurosphere size (μm) and number of DIPG neurospheres treated with the indicated concentrations of PTC-209. CCHMC-DIPG-1 (C-D), CCHMC-DIPG-2 (E-F), and SU-DIPG-XXI (G-H). Error bars represent the standard deviation obtained from triplicates. Each experiment was performed at least twice. *, P

    Techniques Used: Activity Assay, WST-1 Assay, Standard Deviation

    BMI-1 downregulation affects RB pathway, induces G 1 /S cell-cycle arrest and apoptosis in DIPG cells (A) Immunoblot analysis of pRB, total Rb, BMI-1, p21, p16 ink4A and H2AK119Ub extracted from DIPG cell lines treated with DMSO or PTC-209. β-actin and total H2A served as loading controls. (B) Cell cycle analysis of PTC-209 treated DIPG cells. DMSO treatment represents the control. Analysis was performed by gating on live cells only. Percentage of cells in G 1 , S and G 2 /M is indicated. (C) Immunoblot analysis of pH3S10, a marker of mitosis. Total H3 served as loading control. (D) Flow-cytometry analyses of PTC-209 or DMSO treated DIPG cells stained with annexin-V and propidium iodide (PI). The percentage of apoptosis (lower far right quadrant) and overall cell death (upper far right quadrant) is indicated. In all experiments, cells were either treated with DMSO or 5 μM PTC-209 for 3 days.
    Figure Legend Snippet: BMI-1 downregulation affects RB pathway, induces G 1 /S cell-cycle arrest and apoptosis in DIPG cells (A) Immunoblot analysis of pRB, total Rb, BMI-1, p21, p16 ink4A and H2AK119Ub extracted from DIPG cell lines treated with DMSO or PTC-209. β-actin and total H2A served as loading controls. (B) Cell cycle analysis of PTC-209 treated DIPG cells. DMSO treatment represents the control. Analysis was performed by gating on live cells only. Percentage of cells in G 1 , S and G 2 /M is indicated. (C) Immunoblot analysis of pH3S10, a marker of mitosis. Total H3 served as loading control. (D) Flow-cytometry analyses of PTC-209 or DMSO treated DIPG cells stained with annexin-V and propidium iodide (PI). The percentage of apoptosis (lower far right quadrant) and overall cell death (upper far right quadrant) is indicated. In all experiments, cells were either treated with DMSO or 5 μM PTC-209 for 3 days.

    Techniques Used: Cell Cycle Assay, Marker, Flow Cytometry, Cytometry, Staining

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    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    Affinity Purification:

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    Immunofluorescence:

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)
    Article Snippet: .. The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1). .. The secondary HRP-conjugated antibodies for Western blot analysis were purchased from Jackson ImmunoResearch (1:5000–1:30,000), and secondary AlexaFluor-488- or -568-conjugated antibodies for immunofluorescence were from Invitrogen (1:1000–1:2000).

    SDS Page:

    Article Title: TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein
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    Cell Signaling Technology Inc sirt3
    Interaction of NMNAT2 with <t>SIRT3</t> promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P
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    Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the <t>phosphorylated-CDC2</t> and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P
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    Loss of <t>SIRT1</t> catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.
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    Interaction of NMNAT2 with SIRT3 promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 promoted mitotic growth and proliferation of A549 cells. (A) By XTT assays, we investigated the effect of the interaction between NMNAT2 and SIRT3 on cell growth and found that the growth of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in A549 cells (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Interaction of NMNAT2 with SIRT3 increases NAD levels of A549. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in the A549 cells alone. Downregulation of SIRT3, decreased significantly (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 increases NAD levels of A549. NAD level of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was more significant than in the A549 cells alone. Downregulation of SIRT3, decreased significantly (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Map of the NMNAT2 and SIRT3 interaction regions. (A) Mapping of SIRT3 interaction region in NMNAT2. (B) Co-immunoprecipitation of NMNAT2 and SIRT3. Map of the NMNAT2 SIRT3-interacting domains. Myc-SIRT3 and NMNAT2-Flag and its derivatives were overexpressed in A549 cells. NMNAT2-Flag protein was pulled down by Protein G Plus/Protein A Agarose Suspension beads. The presence of SIRT3 was detected by Myc immunoblotting.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Map of the NMNAT2 and SIRT3 interaction regions. (A) Mapping of SIRT3 interaction region in NMNAT2. (B) Co-immunoprecipitation of NMNAT2 and SIRT3. Map of the NMNAT2 SIRT3-interacting domains. Myc-SIRT3 and NMNAT2-Flag and its derivatives were overexpressed in A549 cells. NMNAT2-Flag protein was pulled down by Protein G Plus/Protein A Agarose Suspension beads. The presence of SIRT3 was detected by Myc immunoblotting.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Immunoprecipitation

    Interaction of NMNAT2 with SIRT3 promotes mitotic entry of A549. (A) Interaction of NMNAT2 with SIRT3 did not affect DNA synthesis. A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse-labeled with BrdU (50 μ M) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of the three experiments. (B) Interaction of NMNAT2 with SIRT3 promoted mitotic entry. Cell cycle progression of > 1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. Values are means of three experiments. All data are presented as the mean ± SEM.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 promotes mitotic entry of A549. (A) Interaction of NMNAT2 with SIRT3 did not affect DNA synthesis. A549 cells were transfected with plasmids and synchronized at the G1/S transition as described in Materials and methods. Cells were pulse-labeled with BrdU (50 μ M) for 30 min at indicated time-points after release from the second thymidine block. BrdU positive cells were detected by immunostaining and scored manually. More than 500 cells were counted in each of the three experiments. (B) Interaction of NMNAT2 with SIRT3 promoted mitotic entry. Cell cycle progression of > 1,000 cells was recorded by time-lapse videomicroscopy. The number of mitotic cells was scored by examination of individual cells. Values are means of three experiments. All data are presented as the mean ± SEM.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: DNA Synthesis, Transfection, Labeling, Blocking Assay, Immunostaining

    NMNAT2 interacts with SIRT3 in yeast, in mammalian cells and in vitro . (A) Identification of NMNAT2 as a SIRT3-interacting protein by the yeast two-hybrid assay. Yeast AH109 cells were transformed with different plasmids and grown on SD/-Trp-Leu-His-Ade. +, grown within 96 h; −, no growth within 96 h. Positive colonies were tested for β-galactosidase activity. +, turned blue within 2 h; −, did not turn blue within 2 h. (B) Interaction of NMNAT2 with SIRT3 in mammalian cells. A549 cells, cultured in regular medium, were transfected with expression plasmids as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed using anti-FLAG or anti-Myc monoclonal antibody respectively. (C) To test NMNAT2 interacting with SIRT3 in vivo by Mammalian Two-Hybrid system. A549 cells were transfected with expression plasmids as indicated and cultured in regular medium. At 48 h after transfection, cells were evaluated by firely luciferase assays. Significance was determined on the mean of three different experiments. The luciferase levels of pACT-NMNAT2 and pBIND-SIRT3 were ≥2.64 times higher than negative control (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: NMNAT2 interacts with SIRT3 in yeast, in mammalian cells and in vitro . (A) Identification of NMNAT2 as a SIRT3-interacting protein by the yeast two-hybrid assay. Yeast AH109 cells were transformed with different plasmids and grown on SD/-Trp-Leu-His-Ade. +, grown within 96 h; −, no growth within 96 h. Positive colonies were tested for β-galactosidase activity. +, turned blue within 2 h; −, did not turn blue within 2 h. (B) Interaction of NMNAT2 with SIRT3 in mammalian cells. A549 cells, cultured in regular medium, were transfected with expression plasmids as indicated. Immunoprecipitation (IP) and immunoblotting (IB) were performed using anti-FLAG or anti-Myc monoclonal antibody respectively. (C) To test NMNAT2 interacting with SIRT3 in vivo by Mammalian Two-Hybrid system. A549 cells were transfected with expression plasmids as indicated and cultured in regular medium. At 48 h after transfection, cells were evaluated by firely luciferase assays. Significance was determined on the mean of three different experiments. The luciferase levels of pACT-NMNAT2 and pBIND-SIRT3 were ≥2.64 times higher than negative control (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: In Vitro, Y2H Assay, Transformation Assay, Activity Assay, Cell Culture, Transfection, Expressing, Immunoprecipitation, In Vivo, Luciferase, Negative Control

    Interaction of NMNAT2 with SIRT3 inhibited apoptosis of A549 cells. The result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than in A549 cells (P > 0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 inhibited apoptosis of A549 cells. The result showed that the apoptosis of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 was less than in A549 cells (P > 0.05). The apoptosis of cells were significantly promoted by interfering SIRT3 (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag-NMNAT2 was incubated with PCAF and acetylation of protein was determined by western blotting with antiacetyllysine antibody. (B) Deacetylation of NMNAT2 by SIRT3 in vitro . Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. (C) Quantification of NMNAT2 deacetylation by SIRT3. (D) In vivo deacetylation of NMNAT2 by SIRT3. Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μ M for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by immunoblotting with antiacetyllysine antibody. (E) Quantification of NMNAT2 deacetylation by SIRT3 in vivo . Values are means of three experiments. All data are presented as the mean ± SEM.

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: SIRT3 deacetylates NMNAT2 under in vitro and in vivo assay conditions. (A) In an acetylation buffer Flag-NMNAT2 was incubated with PCAF and acetylation of protein was determined by western blotting with antiacetyllysine antibody. (B) Deacetylation of NMNAT2 by SIRT3 in vitro . Flag-NMNAT2 was acetylated in vitro with PACF and it was precipitated with Flag M2 beads. Acetylated Flag-NMNAT2 was then incubated with beads containing SIRT3 in a deacetylation buffer with or without NAD. SIRT3 was immunoprecipitated from stable A549 cells. (C) Quantification of NMNAT2 deacetylation by SIRT3. (D) In vivo deacetylation of NMNAT2 by SIRT3. Stable cells expressing SIRT3 were induced to overexpress with Flag-NMNAT2 and treated with NAM (10 mM for 24 h) and/or TSA (5 μ M for 6 h) as indicated. Flag-NMNAT2 was immunoprecipitated and the level of acetylation was analyzed by immunoblotting with antiacetyllysine antibody. (E) Quantification of NMNAT2 deacetylation by SIRT3 in vivo . Values are means of three experiments. All data are presented as the mean ± SEM.

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: In Vitro, In Vivo, Incubation, Western Blot, Immunoprecipitation, Expressing

    Interaction of NMNAT2 with SIRT3 boosted strongly mitochondrial functions of A549. Intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compare to A549 cells (P

    Journal: International Journal of Oncology

    Article Title: SIRT3 regulates cell proliferation and apoptosis related to energy metabolism in non-small cell lung cancer cells through deacetylation of NMNAT2

    doi: 10.3892/ijo.2013.2103

    Figure Lengend Snippet: Interaction of NMNAT2 with SIRT3 boosted strongly mitochondrial functions of A549. Intact cellular basal oxygen consumption rates (OCR) of A549 cells were measured by Seahorse XF24 analyzer. The OCR of A549 cells transfected with NMNAT2 or NMNAT2+SIRT3 increased significantly compare to A549 cells (P

    Article Snippet: SIRT3 is the primary mitochondrial deacetylase that modulates mitochondrial metabolic and oxidative stress regulatory pathways ( ).

    Techniques: Transfection

    Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the phosphorylated-CDC2 and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P

    Journal: Molecular Cancer

    Article Title: Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor

    doi: 10.1186/1476-4598-8-34

    Figure Lengend Snippet: Expression changes of Wee1 inhibition signature in xenograft WiDr tumors and their correlation to classical PD marker, pCDC2 . Human WiDr colorectal cancer cells were injected subcutaneously into nude rats. After allowing tumors to establish for 8 days, gencitabine and Wee1 inhibitor were administered in the nude rats as described in the legend of Figure 3. At 8 hr post Wee1 inhibitor administration, mRNA from each WiDr xenograft tumor was applied to quantitative RT-PCR analysis. To correlate the phosphorylated-CDC2 and Wee1 gene expression signature, the level of the pCDC2 normalized to total CDC2 is shown. The correlation coefficient (R) of pCDC2 expression and each mRNA expression is shown in each graph. Data represent mean ± SD. **, P

    Article Snippet: CDC2 protein was solubilized by homogenizing cells in buffer containing 1% NP40, 0.1% Triton X-100, and was detected by Western blotting with an anti-p-CDC2Y15 specific antibody (Cell Signaling #9111).

    Techniques: Expressing, Inhibition, Marker, Injection, Quantitative RT-PCR

    PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Journal: Biochemical Pharmacology

    Article Title: PARP inhibition potentiates the cytotoxic activity of C-1305, a selective inhibitor of topoisomerase II, in human BRCA1-positive breast cancer cells

    doi: 10.1016/j.bcp.2012.07.024

    Figure Lengend Snippet: PARP-1 inhibitor induces site-specific phosphorylation of H2AX and changes in p53 expression in human breast cancer cells. WCLs prepared from untreated control MCF-7 and BT-20 cells and cells exposed to either one inhibitor alone or both inhibitors in tandem were separated on SDS slab gels (10 or 15%) and analyzed by immunoblotting using indicated antibodies as described in detail in Fig. 1 . The intensity of protein bands representing P-Ser139 H2AX and total H2AX protein in each lane was normalized against actin. Then P-Ser139 H2AX/H2AX ratio was calculated and normalized against the ratio calculated for the controls. The relative phosphorylation of H2AX was plotted in the diagram.

    Article Snippet: 2.3 Antibodies The following specific primary antibodies were used to detect the relevant proteins: mouse monoclonal anti-p53 antibody DO-1 and rabbit polyclonal anti-H2AX antibody (from BioLegend, San Diego, CA), anti-BRCA-1 rabbit polyclonal antibody (from Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ER-α rabbit polyclonal antibody (from Sigma–Aldrich, St. Louis, MO), anti-phospho-histone H2AX (Ser139) rabbit polyclonal antibody (from Cell Signaling Technology Inc., Danvers, MA), anti-DBC-1 mouse monoclonal antibody (from Abcam plc, Cambridge, UK), and mouse monoclonal anti-actin antibody (clone C4, ICN Biochemicals, Aurora, OH).

    Techniques: Expressing

    Loss of SIRT1 catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity does not affect metastasis. A ) Mouse lungs displaying metastatic nodules (arrows) (H E, left, scale bar 5 mm, right, scale bar 100 μm) and metastatic nodules stained via immunohistochemistry for polyoma middle T antigen and ERα (scale bar 100 μm) B ) The number of individual metastatic nodules in whole lung H E sections assessed at endpoint. Eight 10µm sections spaced 50µm apart were evaluated in each mouse. N=10 mice per genotype. Points represent individual animals and bars represent the mean number of metastatic nodules. C ) The average number of individual metastatic nodules in whole lung H E sections assessed at endpoint correlated with overall survival time in days.

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Staining, Immunohistochemistry, Mouse Assay

    Abolition of SIRT1 enzymatic activity results in blunted ductal morphogenesis in the mammary gland. Right panel, representative photographs of whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age (scale bar, 5mm). Left panel, a higher magnification view of the ductal network in representative mammary gland whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age. 400X magnification (scale bar, 0.7 mm).

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Abolition of SIRT1 enzymatic activity results in blunted ductal morphogenesis in the mammary gland. Right panel, representative photographs of whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age (scale bar, 5mm). Left panel, a higher magnification view of the ductal network in representative mammary gland whole mounts of the 4 th abdominal mammary gland in Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at eleven weeks of age. 400X magnification (scale bar, 0.7 mm).

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay

    Loss of SIRT1 catalytic activity is associated with increase tumor latency A) Kaplan Meir plot measuring the percentage of mice without any palpable mammary gland mass at the given age. N= 10 mice per genotype. There was a significant delay in the time at which the Sirt1 Y/Y developed their first detectable mass as compared to the Sirt1 +/+ and the Sirt1 Y/+ mice (P

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity is associated with increase tumor latency A) Kaplan Meir plot measuring the percentage of mice without any palpable mammary gland mass at the given age. N= 10 mice per genotype. There was a significant delay in the time at which the Sirt1 Y/Y developed their first detectable mass as compared to the Sirt1 +/+ and the Sirt1 Y/+ mice (P

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay

    Expression of SIRT1, Middle T Antigen, and ERα protein in mammary tumors. Representative immunohistochemical staining for SIRT1 (A-C), Middle T Antigen (D-F), and ERα (G-I) in mammary tumors collected at humane endpoint from Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at 200X magnification (scale bars, 100 μm).

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Expression of SIRT1, Middle T Antigen, and ERα protein in mammary tumors. Representative immunohistochemical staining for SIRT1 (A-C), Middle T Antigen (D-F), and ERα (G-I) in mammary tumors collected at humane endpoint from Sirt1 +/+ , Sirt1 Y/+ and Sirt1 Y/Y mice at 200X magnification (scale bars, 100 μm).

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Expressing, Immunohistochemistry, Staining, Mouse Assay

    Loss of SIRT1 catalytic activity does not affect expression of the PyMT transgene. Representative immunohistochemical staining for Middle T Antigen in mammary glands of PyMT + / Sirt1 +/+ and PyMT + / Sirt1 Y/Y mice collected at 6 weeks of age (scale bars equal to 100 μm). Arrows indicate areas of mammary intraepithelial neoplasia.

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Loss of SIRT1 catalytic activity does not affect expression of the PyMT transgene. Representative immunohistochemical staining for Middle T Antigen in mammary glands of PyMT + / Sirt1 +/+ and PyMT + / Sirt1 Y/Y mice collected at 6 weeks of age (scale bars equal to 100 μm). Arrows indicate areas of mammary intraepithelial neoplasia.

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Expressing, Immunohistochemistry, Staining, Mouse Assay

    Abrogation of SIRT1 catalytic activity does not prevent mammary tumor formation in the MMTV-PyMT mouse model of breast cancer. A ) Kaplan Meir plot showing the percentage of surviving animals over time. N= 10 mice per genotype. Sirt1 Y/Y animals had a significantly longer overall survival time than the Sirt1 +/+ and the Sirt1 Y/+ mice (p

    Journal: PLoS ONE

    Article Title: SIRT1 Catalytic Activity Has Little Effect on Tumor Formation and Metastases in a Mouse Model of Breast Cancer

    doi: 10.1371/journal.pone.0082106

    Figure Lengend Snippet: Abrogation of SIRT1 catalytic activity does not prevent mammary tumor formation in the MMTV-PyMT mouse model of breast cancer. A ) Kaplan Meir plot showing the percentage of surviving animals over time. N= 10 mice per genotype. Sirt1 Y/Y animals had a significantly longer overall survival time than the Sirt1 +/+ and the Sirt1 Y/+ mice (p

    Article Snippet: We undertook to directly test the idea that the protein deacetylase activity of SIRT1 suppresses tumor formation in mice that develop aggressive mammary carcinomas under the influence of the polyoma middle T oncogene.

    Techniques: Activity Assay, Mouse Assay