enzyme linked immunosorbent assay elisa apoptosis assay histone associated dna fragmentation  (Roche)


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    Structured Review

    Roche enzyme linked immunosorbent assay elisa apoptosis assay histone associated dna fragmentation
    Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and <t>apoptosis</t> was estimated using <t>ELISA</t> cell death assay by measuring histone associated <t>DNA</t> fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P
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    1) Product Images from "Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models"

    Article Title: Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models

    Journal: BMC Medicine

    doi: 10.1186/1741-7015-10-80

    Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and apoptosis was estimated using ELISA cell death assay by measuring histone associated DNA fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P
    Figure Legend Snippet: Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and apoptosis was estimated using ELISA cell death assay by measuring histone associated DNA fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P

    Techniques Used: Multiple Displacement Amplification, Expressing, Over Expression, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Sulforhodamine B Assay

    2) Product Images from "Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells"

    Article Title: Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells

    Journal:

    doi: 10.1165/rcmb.2005-0109OC

    PAR-1 agonists induce lung epithelial apoptosis. Apoptosis was assessed using histone-associated DNA fragments (Cell Death Detection assay; A , B , D , E , G , H ) at 4 and 12 h after the treatment with a control peptide (Control) 100 μM, leukocyte
    Figure Legend Snippet: PAR-1 agonists induce lung epithelial apoptosis. Apoptosis was assessed using histone-associated DNA fragments (Cell Death Detection assay; A , B , D , E , G , H ) at 4 and 12 h after the treatment with a control peptide (Control) 100 μM, leukocyte

    Techniques Used: Detection Assay

    PAR-1 siRNA diminishes PAR-1 expression and inhibits leukocyte elastase (LE)-induced apoptosis in BEAS-2B cells. ( A
    Figure Legend Snippet: PAR-1 siRNA diminishes PAR-1 expression and inhibits leukocyte elastase (LE)-induced apoptosis in BEAS-2B cells. ( A

    Techniques Used: Expressing

    Leukocyte elastase (LE) induces lung epithelial apoptosis in a dose-dependent manner. Cell death detection assay ( A–C ) and TUNEL staining ( D–F ) were used for the detection of apoptosis. Three different lung epithelial cell types BEAS-2B
    Figure Legend Snippet: Leukocyte elastase (LE) induces lung epithelial apoptosis in a dose-dependent manner. Cell death detection assay ( A–C ) and TUNEL staining ( D–F ) were used for the detection of apoptosis. Three different lung epithelial cell types BEAS-2B

    Techniques Used: Detection Assay, TUNEL Assay, Staining

    3) Product Images from "Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization"

    Article Title: Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-12-0793

    A, identification of TZs with Rad6 inhibitory activity. In vitro histone H2A ubiquitination assays were conducted as described in Materials and Methods. Rad6B-ubiquitin thioester (Rad6B~Ub) and monoubiquitinated histone H2A (Ub-H2A) were detected by ubiquitin
    Figure Legend Snippet: A, identification of TZs with Rad6 inhibitory activity. In vitro histone H2A ubiquitination assays were conducted as described in Materials and Methods. Rad6B-ubiquitin thioester (Rad6B~Ub) and monoubiquitinated histone H2A (Ub-H2A) were detected by ubiquitin

    Techniques Used: Activity Assay, In Vitro

    4) Product Images from "Protein identification using sequential ion/ion reactions and tandem mass spectrometry"

    Article Title: Protein identification using sequential ion/ion reactions and tandem mass spectrometry

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0503189102

    Online chromatographic separation of large peptides (residues 1–50, ≈½ the protein) from histone H3.1 followed by automated sequential ion/ion reactions (ETD/PTR) and mass spectrometry. ( A ) The tandem mass spectrum generated from
    Figure Legend Snippet: Online chromatographic separation of large peptides (residues 1–50, ≈½ the protein) from histone H3.1 followed by automated sequential ion/ion reactions (ETD/PTR) and mass spectrometry. ( A ) The tandem mass spectrum generated from

    Techniques Used: Mass Spectrometry, Generated

    5) Product Images from "The Yeast Trimeric Guanine Nucleotide-Binding Protein ? Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients"

    Article Title: The Yeast Trimeric Guanine Nucleotide-Binding Protein ? Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

    Journal: Molecular and Cellular Biology

    doi:

    Gpa2p bound to GTP physically interacts with Ime2p in vitro. Purified E. coli Gpa2p-His 6 was preincubated with either GDP (lanes 2 and 4) or GTPγS (lanes 3 and 5) and loaded onto glutathione resin carrying GST (lanes 2 and 3) or GST-Ime2p (lanes 4 and 5). Resin-bound proteins were analyzed by Western blotting with anti-Gpa2p antibodies and by Coomassie blue staining for GST and GST-Ime2p. Lane 1, Gpa2p-His 6 purified from E. coli ; lane M, molecular weight standards.
    Figure Legend Snippet: Gpa2p bound to GTP physically interacts with Ime2p in vitro. Purified E. coli Gpa2p-His 6 was preincubated with either GDP (lanes 2 and 4) or GTPγS (lanes 3 and 5) and loaded onto glutathione resin carrying GST (lanes 2 and 3) or GST-Ime2p (lanes 4 and 5). Resin-bound proteins were analyzed by Western blotting with anti-Gpa2p antibodies and by Coomassie blue staining for GST and GST-Ime2p. Lane 1, Gpa2p-His 6 purified from E. coli ; lane M, molecular weight standards.

    Techniques Used: In Vitro, Purification, Western Blot, Staining, Molecular Weight

    Autoradiograms showing that Gpa2p-His 6 recombinant protein bound to GTPγS inhibits Ime2p kinase activity. (A) Histone H1 phosphorylation was assayed with GST-Ime2p purified from E. coli and [γ- 32 P]ATP in the presence of BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4), washed free of unbound guanine nucleotides. GST was incubated under the same conditions in the presence of BSA (lane 1). As a control, histone H1 phosphorylation by PKA was assayed in the presence of either BSA (lane 5), Gpa2-GDP (lane 6), or Gpa2-GTPγS (lane 7). (B) Ime2p-His 6 purified from MD211 cells transformed with pP583-IME2His6 and grown in SPO medium was assayed for histone H1 phosphorylation in the presence of either BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4). As a control, extracts from yeast cells transformed with an empty plasmid and grown in SPO medium were used (lane 1).
    Figure Legend Snippet: Autoradiograms showing that Gpa2p-His 6 recombinant protein bound to GTPγS inhibits Ime2p kinase activity. (A) Histone H1 phosphorylation was assayed with GST-Ime2p purified from E. coli and [γ- 32 P]ATP in the presence of BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4), washed free of unbound guanine nucleotides. GST was incubated under the same conditions in the presence of BSA (lane 1). As a control, histone H1 phosphorylation by PKA was assayed in the presence of either BSA (lane 5), Gpa2-GDP (lane 6), or Gpa2-GTPγS (lane 7). (B) Ime2p-His 6 purified from MD211 cells transformed with pP583-IME2His6 and grown in SPO medium was assayed for histone H1 phosphorylation in the presence of either BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4). As a control, extracts from yeast cells transformed with an empty plasmid and grown in SPO medium were used (lane 1).

    Techniques Used: Recombinant, Activity Assay, Purification, Incubation, Transformation Assay, Plasmid Preparation

    6) Product Images from "Bovine Herpesvirus 1 Tegument Protein VP22 Interacts with Histones, and the Carboxyl Terminus of VP22 Is Required for Nuclear Localization"

    Article Title: Bovine Herpesvirus 1 Tegument Protein VP22 Interacts with Histones, and the Carboxyl Terminus of VP22 Is Required for Nuclear Localization

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.17.8251-8258.2001

    Antihistone antibody MAB052 recognizes VP22. Whole-cell lysates of MDBK (lane 1), purified BHV-1 (lane 2), and purified His-tagged VP22 were analyzed by immunoblotting and probed with antihistone antibody MAB052 (A) and reprobed with anti-VP22 antibody (B). Note that the slight difference in the band positions of VP22 in lanes 2 and 3 of panels A and B results from the addition of the His tag to VP22 in lane 3.
    Figure Legend Snippet: Antihistone antibody MAB052 recognizes VP22. Whole-cell lysates of MDBK (lane 1), purified BHV-1 (lane 2), and purified His-tagged VP22 were analyzed by immunoblotting and probed with antihistone antibody MAB052 (A) and reprobed with anti-VP22 antibody (B). Note that the slight difference in the band positions of VP22 in lanes 2 and 3 of panels A and B results from the addition of the His tag to VP22 in lane 3.

    Techniques Used: Purification

    Nuclear localization of VP22 is independent of other viral factors. (A) MDBK cells were infected by BHV-1, labeled with anti-VP22, and visualized by indirect immunofluorescence. (B) D17 cells were transfected with full-length VP22 fused with GFP protein. VP22 localizes in cell nuclei in infected or transfected cells.
    Figure Legend Snippet: Nuclear localization of VP22 is independent of other viral factors. (A) MDBK cells were infected by BHV-1, labeled with anti-VP22, and visualized by indirect immunofluorescence. (B) D17 cells were transfected with full-length VP22 fused with GFP protein. VP22 localizes in cell nuclei in infected or transfected cells.

    Techniques Used: Infection, Labeling, Immunofluorescence, Transfection

    Map of the VP22 domain(s) that supports nuclear targeting. (A) Schematic representation of the VP22-GFP constructs used. GFP (shaded boxes) was fused to various domains of VP22 (white boxes) with the start sites and endpoints labeled. D17 cells were transfected with VP22 containing aa 1 to 123 (VP22 1–123 )-GFP (B), VP22 118–258 -GFP (C), VP22 159–258 -GFP (D), VP22 118–221 -GFP (E), VP22 1–197 -GFP (F), VP22 1–221 -GFP (G), or VP22-GFP in the presence of 50 pg of Colcemid per ml (H) and analyzed by fluorescence microscopy directly. Magnification, ×63. Note that the carboxyl terminus of VP22 (C) supports nuclear targeting.
    Figure Legend Snippet: Map of the VP22 domain(s) that supports nuclear targeting. (A) Schematic representation of the VP22-GFP constructs used. GFP (shaded boxes) was fused to various domains of VP22 (white boxes) with the start sites and endpoints labeled. D17 cells were transfected with VP22 containing aa 1 to 123 (VP22 1–123 )-GFP (B), VP22 118–258 -GFP (C), VP22 159–258 -GFP (D), VP22 118–221 -GFP (E), VP22 1–197 -GFP (F), VP22 1–221 -GFP (G), or VP22-GFP in the presence of 50 pg of Colcemid per ml (H) and analyzed by fluorescence microscopy directly. Magnification, ×63. Note that the carboxyl terminus of VP22 (C) supports nuclear targeting.

    Techniques Used: Construct, Labeling, Transfection, Fluorescence, Microscopy

    VP22 interacts with histones. (A) Whole-cell lysate from MDBK cells (lane 1), D17 cells (lane 2), or purified histones at 0.5 mg (lane 3) and 1 mg (lane 4) were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with purified His-tagged VP22 at 4°C overnight, detected with an anti-His tag antibody, and visualized by the ECL method. Histone H1 and histone core proteins (H2A, H2B, H3, and H4) are indicated. (B) Mononucleosomes were purified as described in Materials and Methods. Nucleosomes (200 ng of DNA content) were mixed with 0, 100, 200, 300, 300, and 0 ng of purified VP22 proteins (lanes 1 to 6, respectively) at room temperature for 1 h, with (lanes 5 and 6) or without (lanes 1 to 4) further incubation with anti-VP22 antibody. The formed complexes were analyzed by agarose (0.7%) gel electrophoresis.
    Figure Legend Snippet: VP22 interacts with histones. (A) Whole-cell lysate from MDBK cells (lane 1), D17 cells (lane 2), or purified histones at 0.5 mg (lane 3) and 1 mg (lane 4) were separated by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with purified His-tagged VP22 at 4°C overnight, detected with an anti-His tag antibody, and visualized by the ECL method. Histone H1 and histone core proteins (H2A, H2B, H3, and H4) are indicated. (B) Mononucleosomes were purified as described in Materials and Methods. Nucleosomes (200 ng of DNA content) were mixed with 0, 100, 200, 300, 300, and 0 ng of purified VP22 proteins (lanes 1 to 6, respectively) at room temperature for 1 h, with (lanes 5 and 6) or without (lanes 1 to 4) further incubation with anti-VP22 antibody. The formed complexes were analyzed by agarose (0.7%) gel electrophoresis.

    Techniques Used: Purification, SDS Page, Incubation, Nucleic Acid Electrophoresis

    Acetylation of histone H4 is reduced in VP22-expressing cells. Similar amounts of acid-extracted proteins from VP22-transfected or nontransfected cells were analyzed by immunoblotting and probed with anti-acetyl-histone H4 (Lys 5) (A) and with antihistone H4 (B). Note that the acetylation of histone H4 was decreased in VP22-expressing cells compared to that of control cells.
    Figure Legend Snippet: Acetylation of histone H4 is reduced in VP22-expressing cells. Similar amounts of acid-extracted proteins from VP22-transfected or nontransfected cells were analyzed by immunoblotting and probed with anti-acetyl-histone H4 (Lys 5) (A) and with antihistone H4 (B). Note that the acetylation of histone H4 was decreased in VP22-expressing cells compared to that of control cells.

    Techniques Used: Expressing, Transfection

    Acetylation of histone H4 is reduced in BHV-1- but not in VP22 deletion mutant-infected MDBK cells. Cells were infected with BHV-1 or the VP22 deletion mutant virus (vdUL49) for 18 h and then treated with a 500 nM concentration of the deacetylase inhibitor trichostatin A for 3 h. Acid-soluble protein extracts (20 mg) from mock-infected (lane 1), BHV-1-infected, or vdUL49-infected MDBK cells were fractionated on an SDS-polyacrylamide gel and blotted with anti-acetyl H4 histone, anti-H4, or antiglycoprotein D (gD) antibodies. Note that the acetylation, but not the amount, of histone H4 was reduced in BHV-1- but not vdUL49-infected cells.
    Figure Legend Snippet: Acetylation of histone H4 is reduced in BHV-1- but not in VP22 deletion mutant-infected MDBK cells. Cells were infected with BHV-1 or the VP22 deletion mutant virus (vdUL49) for 18 h and then treated with a 500 nM concentration of the deacetylase inhibitor trichostatin A for 3 h. Acid-soluble protein extracts (20 mg) from mock-infected (lane 1), BHV-1-infected, or vdUL49-infected MDBK cells were fractionated on an SDS-polyacrylamide gel and blotted with anti-acetyl H4 histone, anti-H4, or antiglycoprotein D (gD) antibodies. Note that the acetylation, but not the amount, of histone H4 was reduced in BHV-1- but not vdUL49-infected cells.

    Techniques Used: Mutagenesis, Infection, Concentration Assay, Histone Deacetylase Assay

    7) Product Images from "Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms"

    Article Title: Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.18.6254-6269.2001

    The LMW forms of cyclin E are hyperactive. Cyclin EL-FLAG and cyclin E-FLAG constructs Trunk 1 and Trunk 2 were transfected into two mortal cell strains (81N and 76N), two immortalized cell lines (MCF-10A and 76NE6), and two tumor cell lines (MDA-MB-157 and MDA-MB-436), harvested 16 h posttransfection, and subjected toWestern blot analysis with anti-FLAG antibody, histone H1 and GST-Rb kinase analysis, or immune complex formation with CDK2. For Western blot analysis, 50 μg of protein extract from each condition was analyzed with polyclonal antibody to FLAG. For kinase activity and immune complex formation, equal amounts of protein (250 μg) from cell lysates were prepared from each condition and immunoprecipitated with anti-FLAG antibody (polyclonal) coupled to protein A beads. Histone H1 and GST-Rb were used as substrates in the kinase reaction. For each condition, the resulting autoradiogram of the histone H1 and GST-Rb SDS-PAGE are shown. Immune complex formation with CDK2 was assessed by subjecting the anti-FLAG immunoprecipitates to Western blot analysis using a monoclonal antibody to CDK2.
    Figure Legend Snippet: The LMW forms of cyclin E are hyperactive. Cyclin EL-FLAG and cyclin E-FLAG constructs Trunk 1 and Trunk 2 were transfected into two mortal cell strains (81N and 76N), two immortalized cell lines (MCF-10A and 76NE6), and two tumor cell lines (MDA-MB-157 and MDA-MB-436), harvested 16 h posttransfection, and subjected toWestern blot analysis with anti-FLAG antibody, histone H1 and GST-Rb kinase analysis, or immune complex formation with CDK2. For Western blot analysis, 50 μg of protein extract from each condition was analyzed with polyclonal antibody to FLAG. For kinase activity and immune complex formation, equal amounts of protein (250 μg) from cell lysates were prepared from each condition and immunoprecipitated with anti-FLAG antibody (polyclonal) coupled to protein A beads. Histone H1 and GST-Rb were used as substrates in the kinase reaction. For each condition, the resulting autoradiogram of the histone H1 and GST-Rb SDS-PAGE are shown. Immune complex formation with CDK2 was assessed by subjecting the anti-FLAG immunoprecipitates to Western blot analysis using a monoclonal antibody to CDK2.

    Techniques Used: Construct, Transfection, Multiple Displacement Amplification, Western Blot, Activity Assay, Immunoprecipitation, SDS Page

    The LMW forms of cyclin E bind more effectively to CDK2 than does the full-length form of the protein. Cell lysates were prepared from insect cells coinfected with baculovirus containing the indicated cyclin E constructs and CDK2. (A) At 60 h p.i., equal amounts (50 μg) of protein were added to each lane; the gel was then subjected to Western blot analysis with the indicated antibodies. (B) Histone H1 and GST-Rb kinase assays were also performed on the same cell extracts by immunoprecipitating equal amounts of cell lysate with polyclonal antibody to CDK2 coupled to protein A beads, using histone H1 or GST-Rb as substrates. The autoradiograms of the histone H1 and GST-Rb SDS-PAGE are depicted. (C) Immune complex formation with CDK2 was assessed for the same samples by subjecting the anti-CDK2 immunoprecipitates to Western blot analysis using monoclonal antibodies to CDK2 and FLAG.
    Figure Legend Snippet: The LMW forms of cyclin E bind more effectively to CDK2 than does the full-length form of the protein. Cell lysates were prepared from insect cells coinfected with baculovirus containing the indicated cyclin E constructs and CDK2. (A) At 60 h p.i., equal amounts (50 μg) of protein were added to each lane; the gel was then subjected to Western blot analysis with the indicated antibodies. (B) Histone H1 and GST-Rb kinase assays were also performed on the same cell extracts by immunoprecipitating equal amounts of cell lysate with polyclonal antibody to CDK2 coupled to protein A beads, using histone H1 or GST-Rb as substrates. The autoradiograms of the histone H1 and GST-Rb SDS-PAGE are depicted. (C) Immune complex formation with CDK2 was assessed for the same samples by subjecting the anti-CDK2 immunoprecipitates to Western blot analysis using monoclonal antibodies to CDK2 and FLAG.

    Techniques Used: Construct, Western Blot, SDS Page

    8) Product Images from "Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3"

    Article Title: Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.17.4762

    Fig. 3. VEGFR-2- and VEGFR-3-, but not VEGFR-1-activating ligands inhibit apoptosis of serum-deprived HMVECs. Measurement of the cytoplasmic histone-associated DNA fragments (mono- and oligo nucleosomes) in serum-starved HMVECs consisting of two cell populations of blood vascular and lymphatic endothelial cells ( A ) or in the isolated cell populations after magnetic cell sorting using VEGFR-3 antibodies ( B ). The enrichment factor of cytoplasmic oligonucleosomes in the apoptotic cells grown for 24 h in serum-free medium (BSA) was chosen as 100%. Data represent mean values from three independent experiments (mean ± SD). Grey bars in (B) represent blood vascular endothelial cells and white bars represent VEGFR-3-expressing lymphatic endothelial cells. The inset in (B) shows a northern blot containing 4 µg of total RNAs extracted from blood vascular (–) and lymphatic endothelial cells (+). The blot was probed with radiolabelled cDNA fragments of human VEGFR-2 or VEGFR-3, and the 28S and 18S rRNAs were visualized by ethidium bromide staining of the gel for the control of equal loading. The following concentrations of growth factors were used in the apoptosis assays: bFGF, 10 ng/ml; PlGF, 500 ng/ml; VEGF, 50 ng/ml; VEGF-C, 100 ng/ml; VEGF- C156S, 500 ng/ml; VEGF-D, 500 ng/ml; ORFV2-VEGF, 500 ng/ml; and myelin basic protein (MBP) as an irrelevant control protein, 500 ng/ml.
    Figure Legend Snippet: Fig. 3. VEGFR-2- and VEGFR-3-, but not VEGFR-1-activating ligands inhibit apoptosis of serum-deprived HMVECs. Measurement of the cytoplasmic histone-associated DNA fragments (mono- and oligo nucleosomes) in serum-starved HMVECs consisting of two cell populations of blood vascular and lymphatic endothelial cells ( A ) or in the isolated cell populations after magnetic cell sorting using VEGFR-3 antibodies ( B ). The enrichment factor of cytoplasmic oligonucleosomes in the apoptotic cells grown for 24 h in serum-free medium (BSA) was chosen as 100%. Data represent mean values from three independent experiments (mean ± SD). Grey bars in (B) represent blood vascular endothelial cells and white bars represent VEGFR-3-expressing lymphatic endothelial cells. The inset in (B) shows a northern blot containing 4 µg of total RNAs extracted from blood vascular (–) and lymphatic endothelial cells (+). The blot was probed with radiolabelled cDNA fragments of human VEGFR-2 or VEGFR-3, and the 28S and 18S rRNAs were visualized by ethidium bromide staining of the gel for the control of equal loading. The following concentrations of growth factors were used in the apoptosis assays: bFGF, 10 ng/ml; PlGF, 500 ng/ml; VEGF, 50 ng/ml; VEGF-C, 100 ng/ml; VEGF- C156S, 500 ng/ml; VEGF-D, 500 ng/ml; ORFV2-VEGF, 500 ng/ml; and myelin basic protein (MBP) as an irrelevant control protein, 500 ng/ml.

    Techniques Used: Isolation, FACS, Expressing, Northern Blot, Staining

    9) Product Images from "White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly"

    Article Title: White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly

    Journal:

    doi: 10.1073/pnas.0811233106

    ICP11 binds to host histone proteins. ( A ) Histone H3 and H2A/H2B bind to ICP11. After separation by SDS/PAGE and Coomassie blue staining, the acid-extracted histone proteins from P. monodon hemocyte (lane 1) were identified by LC-MS/MS spectrometry (
    Figure Legend Snippet: ICP11 binds to host histone proteins. ( A ) Histone H3 and H2A/H2B bind to ICP11. After separation by SDS/PAGE and Coomassie blue staining, the acid-extracted histone proteins from P. monodon hemocyte (lane 1) were identified by LC-MS/MS spectrometry (

    Techniques Used: SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    ICP11 colocalizes with histone H3 and γhistone H2A.x in vivo. Control and WSSV-infected (72 hpi) shrimp hemocytes were fixed with paraformaldehyde, stained for ICP11 (FITC, green) and with TRITC (red) for histone H3 ( A and B ) and γhistone
    Figure Legend Snippet: ICP11 colocalizes with histone H3 and γhistone H2A.x in vivo. Control and WSSV-infected (72 hpi) shrimp hemocytes were fixed with paraformaldehyde, stained for ICP11 (FITC, green) and with TRITC (red) for histone H3 ( A and B ) and γhistone

    Techniques Used: In Vivo, Infection, Staining

    10) Product Images from "Dynamic targeting of the replication machinery to sites of DNA damage"

    Article Title: Dynamic targeting of the replication machinery to sites of DNA damage

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200312048

    Visualization of replication factors in the DNA damage response. (A) CDDP induces the relocalization of GFP-PCNA and GFP-RPAp34, but not GFP-histone H2B, into discrete foci within the nucleus of stably expressing Rat-1 cells. Bar, 5 μm. (B) RPAp34 rapidly accumulates into foci in response to UV irradiation. (C) Cells expressing GFP-RPAp34 were exposed to UV irradiation through a porous polycarbonate filter. GFP-RPAp34 only accumulates into foci within the irradiated microdomains 3 μm in diameter. (D) RPAp34 accumulates into foci in response to the stalling of DNA replication with HU or APH. (E) RPAp34 foci appear before the accumulation of H2AX phosphorylation after HU treatment. (F) Camptothecin (CPT) induces the formation of RPAp34 foci that colocalize with γ-H2AX foci. (G) Quantitation of the number of cells with punctate versus diffuse localization of GFP-RPAp34 in asynchronous (left) or serum-starved (right) cultures after various stresses over time. 200 cells were counted at each time point from each of three independent experiments. After 48 h serum-starvation, 4% of the unstressed cells labeled BrdU positive during the 12-h time course of the experiment.
    Figure Legend Snippet: Visualization of replication factors in the DNA damage response. (A) CDDP induces the relocalization of GFP-PCNA and GFP-RPAp34, but not GFP-histone H2B, into discrete foci within the nucleus of stably expressing Rat-1 cells. Bar, 5 μm. (B) RPAp34 rapidly accumulates into foci in response to UV irradiation. (C) Cells expressing GFP-RPAp34 were exposed to UV irradiation through a porous polycarbonate filter. GFP-RPAp34 only accumulates into foci within the irradiated microdomains 3 μm in diameter. (D) RPAp34 accumulates into foci in response to the stalling of DNA replication with HU or APH. (E) RPAp34 foci appear before the accumulation of H2AX phosphorylation after HU treatment. (F) Camptothecin (CPT) induces the formation of RPAp34 foci that colocalize with γ-H2AX foci. (G) Quantitation of the number of cells with punctate versus diffuse localization of GFP-RPAp34 in asynchronous (left) or serum-starved (right) cultures after various stresses over time. 200 cells were counted at each time point from each of three independent experiments. After 48 h serum-starvation, 4% of the unstressed cells labeled BrdU positive during the 12-h time course of the experiment.

    Techniques Used: Stable Transfection, Expressing, Irradiation, Cycling Probe Technology, Quantitation Assay, Labeling

    11) Product Images from "The Drosophila Nuclear Lamina Protein YA Binds to DNA and Histone H2B with Four Domains"

    Article Title: The Drosophila Nuclear Lamina Protein YA Binds to DNA and Histone H2B with Four Domains

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.01-07-0336

    Four Domains in YA Bind to DNA and Histone H2B
    Figure Legend Snippet: Four Domains in YA Bind to DNA and Histone H2B

    Techniques Used:

    YA Binds to Histone H2B
    Figure Legend Snippet: YA Binds to Histone H2B

    Techniques Used:

    Four Domains in YA Bind to DNA and Histone H2B
    Figure Legend Snippet: Four Domains in YA Bind to DNA and Histone H2B

    Techniques Used:

    YA's binding to mitotic chromosomes can be competed by histone H2B. Binding reactions of MBP-HMK-YA with mitotic chromosomes were performed in the presence of 20 μM Drosophila histone H1 (B), equi-molar mix of Drosophila core histones (7 μM each, C), 7 μM of histone H2B (D), or 7 μM of histone H2A (E). Mitotic chromosomes were stained with DAPI (DNA) and anti-MBP antibodies (Anti-MBP). Bar: 10 μm.
    Figure Legend Snippet: YA's binding to mitotic chromosomes can be competed by histone H2B. Binding reactions of MBP-HMK-YA with mitotic chromosomes were performed in the presence of 20 μM Drosophila histone H1 (B), equi-molar mix of Drosophila core histones (7 μM each, C), 7 μM of histone H2B (D), or 7 μM of histone H2A (E). Mitotic chromosomes were stained with DAPI (DNA) and anti-MBP antibodies (Anti-MBP). Bar: 10 μm.

    Techniques Used: Binding Assay, Staining

    12) Product Images from "Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin"

    Article Title: Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm032

    Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.
    Figure Legend Snippet: Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.

    Techniques Used: Activity Assay, Transformation Assay, Incubation

    Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.
    Figure Legend Snippet: Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.

    Techniques Used: Modification, Agarose Gel Electrophoresis, Incubation

    Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.
    Figure Legend Snippet: Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.

    Techniques Used: Sequencing, Modification, Nucleic Acid Electrophoresis, Incubation

    13) Product Images from "Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin"

    Article Title: Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm032

    Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.
    Figure Legend Snippet: Primer extension activity of RT HIV-1 using the 17mer/44mer primer/template duplex. The experiments were conducted for the times indicated in the figure (5–90 min) using undamaged templates (lanes 2–5), the template containing single, site-specific 1,2-GG intrastrand CL of cisplatin (lanes 6–9) and the template containing single DPCL formed by the transformation of the template containing site-specific 1,2-GG intrastrand CL of cisplatin incubated with histone H1 (lanes 10–13). Lane 1, 17-mer primer. The pause sites opposite the platinated guanines and the nucleotide preceding the platinated guanines (thymine residue on the 3′ side of the CL) are marked 34, 33, 32, respectively. The nucleotide sequences of the templates and the primers are shown beneath the gels. See the text for other details.

    Techniques Used: Activity Assay, Transformation Assay, Incubation

    Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.
    Figure Legend Snippet: Formation of DPCLs of unmodified and platinated 213-bp DNA fragment globally modified by cisplatin or transplatin ( r b = 0.025) with KF − and histone H1 assessed by agarose gel electrophoresis; the fragment was incubated with the protein for 24 h. Lanes: 1, 2, the fragment modified by cisplatin incubated with KF − , histone H1, respectively; 3, 4, the fragment modified by cisplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 5, 6, the fragment modified by transplatin incubated with KF − , histone H1, respectively; 7, 8, the fragment modified by transplatin incubated in the buffer used for reaction with KF − or histone H1, respectively, no protein added; 9, 10, the unplatinated fragment incubated with KF − or histone H1, respectively. See the text for other details.

    Techniques Used: Modification, Agarose Gel Electrophoresis, Incubation

    Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.
    Figure Legend Snippet: Formation of DPCLs of unmodified and platinated oligodeoxyribonucleotide duplexes 40 bp (A and B) or NF-κB ( 20 ) (C) (see Figure 1 B for their nucleotide sequence) globally modified by cisplatin or transplatin ( r b = 0.025) with KF − (A), histone H1 (B) and NF-κB (C) assessed by SDS/PAA gel electrophoresis. Lanes: 1–3, the duplex modified by cisplatin incubated with the protein for 1, 4 and 24 h, respectively; 4–6, the duplex modified by transplatin incubated with the protein for 1, 4 and 24 h, respectively; 7, control, unplatinated duplex, no protein added; 8, control, unplatinated duplex incubated with the protein for 24 h; 9, control, duplex modified by cisplatin ( r b = 0.025), no protein added; 10, control, duplex modified by cisplatin ( r b = 0.025) incubated with the protein for 24 h. See the text for other details.

    Techniques Used: Sequencing, Modification, Nucleic Acid Electrophoresis, Incubation

    14) Product Images from "Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul"

    Article Title: Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-203

    Immunoblotting for β-Catenin using lysates from MCF-7 (A) or MDA-MB-231 (C) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA–transfected cells. Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (B) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P
    Figure Legend Snippet: Immunoblotting for β-Catenin using lysates from MCF-7 (A) or MDA-MB-231 (C) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA–transfected cells. Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (B) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P

    Techniques Used: Multiple Displacement Amplification, Transfection

    Immunoblotting for TCF-4 using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 μmol/L GL for 24 h. Immunoblotting for TCF-4 using lysates from MCF-7 (C) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or TCF-4 -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated control cells (A-B) or relative to DMSO-treated nonspecific control siRNA–transfected cells (C-D) . Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (E) or MDA-MB-231 (F) cells transiently transfected with a control nonspecific siRNA or TCF-4-targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P
    Figure Legend Snippet: Immunoblotting for TCF-4 using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 μmol/L GL for 24 h. Immunoblotting for TCF-4 using lysates from MCF-7 (C) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or TCF-4 -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated control cells (A-B) or relative to DMSO-treated nonspecific control siRNA–transfected cells (C-D) . Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (E) or MDA-MB-231 (F) cells transiently transfected with a control nonspecific siRNA or TCF-4-targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P

    Techniques Used: Multiple Displacement Amplification, Transfection

    15) Product Images from "Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings"

    Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9969

    ODE activates p53 signaling in CRC cells HCT-116 cells were treated with or without ODE at applied concentrations, cells were further cultured, expressions of listed proteins were tested by Western blots A and C ., the association between AMPKα1 (regular and p-) and p53 (regular and p-) was examined by co-immunoprecipitation (“Co-IP”) assay B ., IgG was also included as a Co-IP control (B). Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with applied ODE, p53 (regular and p-) and Tubulin expressions were tested by Western blots D. Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”) or p53-shRNA (“−1/−-2”) as well as their parental cells were treated with applied ODE, cell viability (MTT assay, E. ) and cell apoptosis (Histone DNA ELISA assay, F. ) were tested, expression of p53 in these cells was also shown (F, upper panel). p53 (regular and p-) and Tubulin expressions in ODE (50 μg/mL)-treated primary CRC cells (patient-1 derived) were shown G . p53 (regular and p-) and AMPKα1 expressions in ODE (50 μg/mL)-treated primary CRC cells with scramble control siRNA (“scr-siRNA”) or AMPKα1 siRNA (“−1/−2”) were shown H. Kinase phosphorylations and p53 expression were quantified. Data in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: ODE activates p53 signaling in CRC cells HCT-116 cells were treated with or without ODE at applied concentrations, cells were further cultured, expressions of listed proteins were tested by Western blots A and C ., the association between AMPKα1 (regular and p-) and p53 (regular and p-) was examined by co-immunoprecipitation (“Co-IP”) assay B ., IgG was also included as a Co-IP control (B). Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) were treated with applied ODE, p53 (regular and p-) and Tubulin expressions were tested by Western blots D. Stable HCT-116 cells expressing scramble-shRNA (“scr-shRNA”) or p53-shRNA (“−1/−-2”) as well as their parental cells were treated with applied ODE, cell viability (MTT assay, E. ) and cell apoptosis (Histone DNA ELISA assay, F. ) were tested, expression of p53 in these cells was also shown (F, upper panel). p53 (regular and p-) and Tubulin expressions in ODE (50 μg/mL)-treated primary CRC cells (patient-1 derived) were shown G . p53 (regular and p-) and AMPKα1 expressions in ODE (50 μg/mL)-treated primary CRC cells with scramble control siRNA (“scr-siRNA”) or AMPKα1 siRNA (“−1/−2”) were shown H. Kinase phosphorylations and p53 expression were quantified. Data in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Cell Culture, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, shRNA, Dominant Negative Mutation, MTT Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

    AMPK activation is required for ODE-induced anti-CRC cell activity Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) as well as their parental cells were treated with or without applied ODE, cells were further cultured, cell viability ( A. , MTT assay), cell death ( B. , trypan blue staining assay) and cell apoptosis ( C. , Histone DNA ELISA assay) were tested. Primary CRC cells (patient-1-dervied), transfected with scramble control siRNA (“scr-siRNA”) or AMPKα1-siRNA (−1/−2), were treated with ODE for indicated time, expressions of listed proteins were shown D ., cell viability E . and apoptosis F . were tested similarly. Kinase phosphorylations were quantified (D). Data in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: AMPK activation is required for ODE-induced anti-CRC cell activity Stable HCT-116 cells expressing scramble control shRNA (“scr-shRNA”), AMPKα1-shRNA or dominant negative (dn)-AMPKα1 (“dnAMPKα1”) as well as their parental cells were treated with or without applied ODE, cells were further cultured, cell viability ( A. , MTT assay), cell death ( B. , trypan blue staining assay) and cell apoptosis ( C. , Histone DNA ELISA assay) were tested. Primary CRC cells (patient-1-dervied), transfected with scramble control siRNA (“scr-siRNA”) or AMPKα1-siRNA (−1/−2), were treated with ODE for indicated time, expressions of listed proteins were shown D ., cell viability E . and apoptosis F . were tested similarly. Kinase phosphorylations were quantified (D). Data in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Activation Assay, Activity Assay, Expressing, shRNA, Dominant Negative Mutation, Cell Culture, MTT Assay, Staining, Enzyme-linked Immunosorbent Assay, Transfection

    16) Product Images from "The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability"

    Article Title: The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky008

    FKBP25 is required for timely progression through the cell cycle. ( A ) Western blot showing FKBP25 knockdown in U2OS cells transfected with siRNAs. GAPDH is shown as a loading control. ( B ) MTT proliferation assay measuring cell viability 72 h post-knockdown. Results shown are the mean ± SD of six independent transfections plated at three densities. ( C – E ) Cell cycle analysis of FKBP25 knockdown cells by flow-cytometry. (C) Histogram of PI-stained cells depicting cell cycle distribution, inlay bar graphs depict the mean ±SD of three replicates. (D) Dot-plot representing fluorescence intensity of H3pS10 levels versus PI-stain. (E) Quantification of H3pS10 positive mitotic cells as a percent of G2/M cells in D. Shown is the mean ±SD for three replicates. ( F ) Schematic representation of nocodazole trap; cells incubated for 8 h in nocodazole to halt progression into G1 of the cell cycle. ( G – I ) Flow cytometry analysis of 8 h nocodazole-trapped cells. (G) Dot-plot of H3pS10 expression versus PI-staining. (H) Quantification of cells remaining in G1. Results are shown as the mean ±SD of three replicates. (I) Percentage of G2/M cells in mitosis. Results are presented as the mean ±SD of three replicates. ( J ) Western blot analysis of mitotic entry 8 h post-nocodazole trap of stable FKBP25 shRNA knockdown U2OS cells. Histone H3 and α-tubulin are shown as loading controls. ( K ) Schematic representation of synchronization at G2/M border by thymidine block followed by CDK1 inhibition (RO3306). ( L ) Western blot analysis of mitotic entry in siRNA transfected cells released from G2/M transition block. ( M ) Flow cytometry analysis of PI-stained stable shRNA FKBP25 knockdown cells released from G2/M transition block.
    Figure Legend Snippet: FKBP25 is required for timely progression through the cell cycle. ( A ) Western blot showing FKBP25 knockdown in U2OS cells transfected with siRNAs. GAPDH is shown as a loading control. ( B ) MTT proliferation assay measuring cell viability 72 h post-knockdown. Results shown are the mean ± SD of six independent transfections plated at three densities. ( C – E ) Cell cycle analysis of FKBP25 knockdown cells by flow-cytometry. (C) Histogram of PI-stained cells depicting cell cycle distribution, inlay bar graphs depict the mean ±SD of three replicates. (D) Dot-plot representing fluorescence intensity of H3pS10 levels versus PI-stain. (E) Quantification of H3pS10 positive mitotic cells as a percent of G2/M cells in D. Shown is the mean ±SD for three replicates. ( F ) Schematic representation of nocodazole trap; cells incubated for 8 h in nocodazole to halt progression into G1 of the cell cycle. ( G – I ) Flow cytometry analysis of 8 h nocodazole-trapped cells. (G) Dot-plot of H3pS10 expression versus PI-staining. (H) Quantification of cells remaining in G1. Results are shown as the mean ±SD of three replicates. (I) Percentage of G2/M cells in mitosis. Results are presented as the mean ±SD of three replicates. ( J ) Western blot analysis of mitotic entry 8 h post-nocodazole trap of stable FKBP25 shRNA knockdown U2OS cells. Histone H3 and α-tubulin are shown as loading controls. ( K ) Schematic representation of synchronization at G2/M border by thymidine block followed by CDK1 inhibition (RO3306). ( L ) Western blot analysis of mitotic entry in siRNA transfected cells released from G2/M transition block. ( M ) Flow cytometry analysis of PI-stained stable shRNA FKBP25 knockdown cells released from G2/M transition block.

    Techniques Used: Western Blot, Transfection, MTT Assay, Proliferation Assay, Cell Cycle Assay, Flow Cytometry, Cytometry, Staining, Fluorescence, Incubation, Expressing, shRNA, Blocking Assay, Inhibition

    FKBP25 is phosphorylated by PKC. ( A – D ) In vitro kinase assays. (A) Western blot analysis of recombinant histone H3 alone, incubated with mitotic extract, or incubated with mitotic extract and the Aurora kinase inhibitor MK-0457 (50 μM) in kinase buffer. (B) Purified full-length proteins were incubated with mitotic extract in the presence of γ[ 32 P]-ATP, resolved by SDS-PAGE and visualized by autoradiography. For the Cntrl lane, no recombinant protein included in the reaction. Histone H3 included as a positive control. (C) Identification of putative mitotic kinase by in vitro kinase assay in the presence of mitotic kinase inhibitors. Assays performed as in B with the inclusion of the kinase inhibitors staurosporine (broad range), Gö 6983 (PKC), Tyrphostin AG1112 (CKII), Roscovitine (CDKs), RO3306 (CDK1) and MK-0457(Aurora). (D) In vitro kinase assay with recombinant canonical PKC kinases and CDK1. Recombinant FKBP25 incubated with purified PKCα (12.5 ng), PKCβII (12.5 ng) or CDK1-cyclinB1 (20 ng). ( E ) Schematic representation of mass spectrometry identified phosphoresidues on recombinant FKBP25 in vitro phosphorylated with either PKCα or PKCβII.
    Figure Legend Snippet: FKBP25 is phosphorylated by PKC. ( A – D ) In vitro kinase assays. (A) Western blot analysis of recombinant histone H3 alone, incubated with mitotic extract, or incubated with mitotic extract and the Aurora kinase inhibitor MK-0457 (50 μM) in kinase buffer. (B) Purified full-length proteins were incubated with mitotic extract in the presence of γ[ 32 P]-ATP, resolved by SDS-PAGE and visualized by autoradiography. For the Cntrl lane, no recombinant protein included in the reaction. Histone H3 included as a positive control. (C) Identification of putative mitotic kinase by in vitro kinase assay in the presence of mitotic kinase inhibitors. Assays performed as in B with the inclusion of the kinase inhibitors staurosporine (broad range), Gö 6983 (PKC), Tyrphostin AG1112 (CKII), Roscovitine (CDKs), RO3306 (CDK1) and MK-0457(Aurora). (D) In vitro kinase assay with recombinant canonical PKC kinases and CDK1. Recombinant FKBP25 incubated with purified PKCα (12.5 ng), PKCβII (12.5 ng) or CDK1-cyclinB1 (20 ng). ( E ) Schematic representation of mass spectrometry identified phosphoresidues on recombinant FKBP25 in vitro phosphorylated with either PKCα or PKCβII.

    Techniques Used: In Vitro, Western Blot, Recombinant, Incubation, Purification, SDS Page, Autoradiography, Positive Control, Kinase Assay, Mass Spectrometry

    17) Product Images from "The role of cdc2 in the expression of herpes simplex virus genes"

    Article Title: The role of cdc2 in the expression of herpes simplex virus genes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    ( A ) Immunoblot of electrophoretically separated lysates of HeLa cells reacted with antibody to HA tag. HeLa cells were mock transfected or transfected with cytomegalovirus expression plasmids carrying HA-tagged dominant-negative cdc2 (pCMVcdc2-dn). The cells were harvested 36 h after transfection, solubilized, electrophoretically separated on 10% bisacrylamide gels, and probed with antibody to HA. ( B ) Autoradiographic image of histone H1 phosphorylated in vitro by immunoprecipitated cdc2. HeLa cells were infected with HSV-1(F) 36 h after transfection with pCMVcdc2-dn and incubated for an additional 14 h at 37°C. Histone H1 was mixed with immune-precipitated Cdc2 from harvested, lysed cells. The reaction mixtures were separated on denaturing gels as described in Materials and Methods .
    Figure Legend Snippet: ( A ) Immunoblot of electrophoretically separated lysates of HeLa cells reacted with antibody to HA tag. HeLa cells were mock transfected or transfected with cytomegalovirus expression plasmids carrying HA-tagged dominant-negative cdc2 (pCMVcdc2-dn). The cells were harvested 36 h after transfection, solubilized, electrophoretically separated on 10% bisacrylamide gels, and probed with antibody to HA. ( B ) Autoradiographic image of histone H1 phosphorylated in vitro by immunoprecipitated cdc2. HeLa cells were infected with HSV-1(F) 36 h after transfection with pCMVcdc2-dn and incubated for an additional 14 h at 37°C. Histone H1 was mixed with immune-precipitated Cdc2 from harvested, lysed cells. The reaction mixtures were separated on denaturing gels as described in Materials and Methods .

    Techniques Used: Transfection, Expressing, Dominant Negative Mutation, In Vitro, Immunoprecipitation, Infection, Incubation

    ( A ) Coomassie blue staining of GST fusion proteins. GST alone, GST-ICP0 20–241, GST-ICP0 543–768, and U L 34 1–240 were purified with the aid of glutathione-agarose beads, electrophoretically separated on 10% bisacrylamide gels, and stained with Coomassie blue. ( B ) Autoradiographic image of cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Cdc2 was immunoprecipitated from uninfected HeLa cells with cdc2 antibody, and histone H1 or GST fusion proteins were tested as substrates for cdc2. Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film.
    Figure Legend Snippet: ( A ) Coomassie blue staining of GST fusion proteins. GST alone, GST-ICP0 20–241, GST-ICP0 543–768, and U L 34 1–240 were purified with the aid of glutathione-agarose beads, electrophoretically separated on 10% bisacrylamide gels, and stained with Coomassie blue. ( B ) Autoradiographic image of cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Cdc2 was immunoprecipitated from uninfected HeLa cells with cdc2 antibody, and histone H1 or GST fusion proteins were tested as substrates for cdc2. Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film.

    Techniques Used: Staining, Purification, Immunoprecipitation

    ( A ) Autoradiographic image of nocodazole-induced cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Replicate HeLa cell cultures were mock-treated or treated with nocodazole for 20 h. cdc2 was immunoprecipitated from lysates and reacted with indicated substrates. Reaction mixtures were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. ( B ) Autoradiographic image of electrophoretically separated histone H1 or GST-ICP0 20–241 polypeptide reacted with immune-precipitated cdc2 in the presence of various concentrations of roscovitine. The cdc2 kinase was immune-precipitated from HEp-2 cells treated with nocodazole for 20 h and reacted with roscovitine before the addition of complete kinase buffer and substrates as described in Materials and Methods . Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. ( C ) Quantification of the amount of radioactivity in substrates phosphorylated by cdc2 in the presence or absence of roscovitine. Radioactivity in each band shown in B was quantified with the aid of Storm 860 PhosphorImager. The radioactivity in each band was normalized with respect to that of untreated (no roscovitine) reaction mixtures (100%). The dashed line represents 50% reduction in phosphorylation.
    Figure Legend Snippet: ( A ) Autoradiographic image of nocodazole-induced cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Replicate HeLa cell cultures were mock-treated or treated with nocodazole for 20 h. cdc2 was immunoprecipitated from lysates and reacted with indicated substrates. Reaction mixtures were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. ( B ) Autoradiographic image of electrophoretically separated histone H1 or GST-ICP0 20–241 polypeptide reacted with immune-precipitated cdc2 in the presence of various concentrations of roscovitine. The cdc2 kinase was immune-precipitated from HEp-2 cells treated with nocodazole for 20 h and reacted with roscovitine before the addition of complete kinase buffer and substrates as described in Materials and Methods . Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. ( C ) Quantification of the amount of radioactivity in substrates phosphorylated by cdc2 in the presence or absence of roscovitine. Radioactivity in each band shown in B was quantified with the aid of Storm 860 PhosphorImager. The radioactivity in each band was normalized with respect to that of untreated (no roscovitine) reaction mixtures (100%). The dashed line represents 50% reduction in phosphorylation.

    Techniques Used: Immunoprecipitation, Radioactivity

    18) Product Images from "The Yeast Trimeric Guanine Nucleotide-Binding Protein ? Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients"

    Article Title: The Yeast Trimeric Guanine Nucleotide-Binding Protein ? Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients

    Journal: Molecular and Cellular Biology

    doi:

    Autoradiograms showing that Gpa2p-His 6 recombinant protein bound to GTPγS inhibits Ime2p kinase activity. (A) Histone H1 phosphorylation was assayed with GST-Ime2p purified from E. coli and [γ- 32 P]ATP in the presence of BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4), washed free of unbound guanine nucleotides. GST was incubated under the same conditions in the presence of BSA (lane 1). As a control, histone H1 phosphorylation by PKA was assayed in the presence of either BSA (lane 5), Gpa2-GDP (lane 6), or Gpa2-GTPγS (lane 7). (B) Ime2p-His 6 purified from MD211 cells transformed with pP583-IME2His6 and grown in SPO medium was assayed for histone H1 phosphorylation in the presence of either BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4). As a control, extracts from yeast cells transformed with an empty plasmid and grown in SPO medium were used (lane 1).
    Figure Legend Snippet: Autoradiograms showing that Gpa2p-His 6 recombinant protein bound to GTPγS inhibits Ime2p kinase activity. (A) Histone H1 phosphorylation was assayed with GST-Ime2p purified from E. coli and [γ- 32 P]ATP in the presence of BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4), washed free of unbound guanine nucleotides. GST was incubated under the same conditions in the presence of BSA (lane 1). As a control, histone H1 phosphorylation by PKA was assayed in the presence of either BSA (lane 5), Gpa2-GDP (lane 6), or Gpa2-GTPγS (lane 7). (B) Ime2p-His 6 purified from MD211 cells transformed with pP583-IME2His6 and grown in SPO medium was assayed for histone H1 phosphorylation in the presence of either BSA (lane 2), Gpa2p bound to GDP (lane 3), or Gpa2p bound to GTPγS (lane 4). As a control, extracts from yeast cells transformed with an empty plasmid and grown in SPO medium were used (lane 1).

    Techniques Used: Recombinant, Activity Assay, Purification, Incubation, Transformation Assay, Plasmid Preparation

    19) Product Images from "Timeless Links Replication Termination to Mitotic Kinase Activation"

    Article Title: Timeless Links Replication Termination to Mitotic Kinase Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019596

    Cell cycle dependent interactions between Tim and mitotic entry kinases. A–B) siControl and siTim transfected HCT116 cells were synchronized by double thymidine and then assayed by PI staining and FACS analysis for cell cycle profile. C) Western blot of total cell extracts of synchronize HCT116 cells after siControl or siTim at cell stages shown in panels A and B for 3, 6, 9, and 12 hrs post-release from thymidine block. Western blot with antibody to Tim, Plk1, Aurora A1, Aurora B1, Cyclin B1, CDK1, Cdh1, Cdc25, histone H3 phospho S10, hitone H3, or Actin, are indicated. D) Immunoprecipitation with anti-Tim or control IgG antibody with extracts from HCT116 cells at 0, 2, 4, 6, or 8 hrs post-arrest from thymidine block. IPs were assayed by Western immunoblot (IB) with Tim, Plk1 Aurora A, or Aurora B antibody as indicated. E) HCT116 cells were synchronized as in panel D, and extracts were subject to IP with antibody to Plk1 (middle panel) or Aurora A (lower panel), followed by Western blot with anti-Tim. Input is shown in top panel, as indicated.
    Figure Legend Snippet: Cell cycle dependent interactions between Tim and mitotic entry kinases. A–B) siControl and siTim transfected HCT116 cells were synchronized by double thymidine and then assayed by PI staining and FACS analysis for cell cycle profile. C) Western blot of total cell extracts of synchronize HCT116 cells after siControl or siTim at cell stages shown in panels A and B for 3, 6, 9, and 12 hrs post-release from thymidine block. Western blot with antibody to Tim, Plk1, Aurora A1, Aurora B1, Cyclin B1, CDK1, Cdh1, Cdc25, histone H3 phospho S10, hitone H3, or Actin, are indicated. D) Immunoprecipitation with anti-Tim or control IgG antibody with extracts from HCT116 cells at 0, 2, 4, 6, or 8 hrs post-arrest from thymidine block. IPs were assayed by Western immunoblot (IB) with Tim, Plk1 Aurora A, or Aurora B antibody as indicated. E) HCT116 cells were synchronized as in panel D, and extracts were subject to IP with antibody to Plk1 (middle panel) or Aurora A (lower panel), followed by Western blot with anti-Tim. Input is shown in top panel, as indicated.

    Techniques Used: Transfection, Staining, FACS, Western Blot, Blocking Assay, Immunoprecipitation

    Tim is required for Plk1 and Aurora A kinase activity in vitro. A–B) HCT116 cells were transfected with siControl (odd lanes) or siTim (even lanes) and then subject to IP with IgG (lanes 1–4, and 13, and 14), Plk1 (lanes 5–8), Aurora A (lanes 9–12), or Aurora B (lanes 15 and 16). Purified histone H3 substrated was added to lanes 3,4, 7,8, 11, 12, 13–16, and the IPs were incubated with 32 P-γATP under kinase conditions for 30 min. Coomassie blue stain of SDS-PAGE containg the kinase reaction is shown in panel A. The autoradiogram of the 32 P-histone H3 is shown in panel B and indicated by the arrow. C) Western blot of IP material used for kinase reactions for Plk1, Aurora A, and Aurora B, as indicated.
    Figure Legend Snippet: Tim is required for Plk1 and Aurora A kinase activity in vitro. A–B) HCT116 cells were transfected with siControl (odd lanes) or siTim (even lanes) and then subject to IP with IgG (lanes 1–4, and 13, and 14), Plk1 (lanes 5–8), Aurora A (lanes 9–12), or Aurora B (lanes 15 and 16). Purified histone H3 substrated was added to lanes 3,4, 7,8, 11, 12, 13–16, and the IPs were incubated with 32 P-γATP under kinase conditions for 30 min. Coomassie blue stain of SDS-PAGE containg the kinase reaction is shown in panel A. The autoradiogram of the 32 P-histone H3 is shown in panel B and indicated by the arrow. C) Western blot of IP material used for kinase reactions for Plk1, Aurora A, and Aurora B, as indicated.

    Techniques Used: Activity Assay, In Vitro, Transfection, Purification, Incubation, Staining, SDS Page, Western Blot

    20) Product Images from "8-bromo-7-methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin-sensitive and resistant ovarian cancer cells"

    Article Title: 8-bromo-7-methoxychrysin induces apoptosis by regulating Akt/FOXO3a pathway in cisplatin-sensitive and resistant ovarian cancer cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2015.4039

    BrMC induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells. (A and B) Effect of cisplatin on apoptosis in ovarian cancer cell lines in a dose-dependent manner. Cells were treated with the indicated concentrations of cisplatin for 48 h. ELISA was used to determine histone/DNA fragmentation. Data are presented as the mean ± SD (n=4). * P
    Figure Legend Snippet: BrMC induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells. (A and B) Effect of cisplatin on apoptosis in ovarian cancer cell lines in a dose-dependent manner. Cells were treated with the indicated concentrations of cisplatin for 48 h. ELISA was used to determine histone/DNA fragmentation. Data are presented as the mean ± SD (n=4). * P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    21) Product Images from "Extracellular histones are major mediators of death in sepsis"

    Article Title: Extracellular histones are major mediators of death in sepsis

    Journal: Nature medicine

    doi: 10.1038/nm.2053

    Cytotoxicity of extracellular histones toward endothelium and APC cleavage of histones. (a) EA.hy926 cells were cultured with calf thymus histones (50µg ml −1 ) or calf thymus histone H1, H2A, H2B, H3 or H4 (20µg ml −1 ) for 1 hr at 37°C. Cell damage was measured by flow cytometry for PI staining. (b) APC (100 nM) was absent or present during the incubations with histones, H3 or H4 in the above assays. (c) SDS–PAGE analysis of purified calf thymus H3 (top panel) or H4 (bottom panel) (100 µg ml −1 ) incubated with the indicated concentrations of human APC for 1 hr at 37°C. (d) SDS–PAGE analysis of purified calf thymus histone H3 (top panel) or H4 (bottom panel) (100 µg ml −1 ) incubated with 10 nM human APC in the absence or presence of 0.5 mg ml −1 PS/PC or PE/PS/PC liposomes for 1 hr at 37°C.
    Figure Legend Snippet: Cytotoxicity of extracellular histones toward endothelium and APC cleavage of histones. (a) EA.hy926 cells were cultured with calf thymus histones (50µg ml −1 ) or calf thymus histone H1, H2A, H2B, H3 or H4 (20µg ml −1 ) for 1 hr at 37°C. Cell damage was measured by flow cytometry for PI staining. (b) APC (100 nM) was absent or present during the incubations with histones, H3 or H4 in the above assays. (c) SDS–PAGE analysis of purified calf thymus H3 (top panel) or H4 (bottom panel) (100 µg ml −1 ) incubated with the indicated concentrations of human APC for 1 hr at 37°C. (d) SDS–PAGE analysis of purified calf thymus histone H3 (top panel) or H4 (bottom panel) (100 µg ml −1 ) incubated with 10 nM human APC in the absence or presence of 0.5 mg ml −1 PS/PC or PE/PS/PC liposomes for 1 hr at 37°C.

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Staining, SDS Page, Purification, Incubation

    APC cleaves histones both in vitro and in vivo . (a) EA.hy926 cells were cultured with calf thymus histones in the indicated concentration in the absence or presence of APC (10 or 100 nM) at 37°C for 1 hr. Cell damage was measured by flow cytometry for PI staining and expressed as mean fluorescence index (MFI). (b) Calf thymus histones (50 µg ml −1 ) incubated with APC (100 nM) at 37°C for the indicated time and then mixed with PPACK (10 µM) to inactivate APC. The above medium was used to culture EA.hy926 cells for 1 hr for cytotoxicity assay or (c) subjected to SDS–PAGE and Western blotting for H3 or H4 at the times indicated. (d) EA.hy926 cells were cultured with calf thymus histones (50µg ml −1 ) in the absence or presence of protein C (100 nM), thrombin (T) (10 nM) or APC (100 nM) at 37°C for 30 min. Cell damage was measured by flow cytometry for PI staining. (e) Western blot analysis for H3 of baboon plasma samples at the times indicated after E. coli or E. coli plus APC challenge. (f) Western blot analysis for H3 of plasma samples taken at the times indicated from the start of APC treatment of a human septic patient.
    Figure Legend Snippet: APC cleaves histones both in vitro and in vivo . (a) EA.hy926 cells were cultured with calf thymus histones in the indicated concentration in the absence or presence of APC (10 or 100 nM) at 37°C for 1 hr. Cell damage was measured by flow cytometry for PI staining and expressed as mean fluorescence index (MFI). (b) Calf thymus histones (50 µg ml −1 ) incubated with APC (100 nM) at 37°C for the indicated time and then mixed with PPACK (10 µM) to inactivate APC. The above medium was used to culture EA.hy926 cells for 1 hr for cytotoxicity assay or (c) subjected to SDS–PAGE and Western blotting for H3 or H4 at the times indicated. (d) EA.hy926 cells were cultured with calf thymus histones (50µg ml −1 ) in the absence or presence of protein C (100 nM), thrombin (T) (10 nM) or APC (100 nM) at 37°C for 30 min. Cell damage was measured by flow cytometry for PI staining. (e) Western blot analysis for H3 of baboon plasma samples at the times indicated after E. coli or E. coli plus APC challenge. (f) Western blot analysis for H3 of plasma samples taken at the times indicated from the start of APC treatment of a human septic patient.

    Techniques Used: In Vitro, In Vivo, Cell Culture, Concentration Assay, Flow Cytometry, Cytometry, Staining, Fluorescence, Incubation, Cytotoxicity Assay, SDS Page, Western Blot

    Intravenous injection of histones elicits inflammatory and cell injury responses. (a) Survival rates of mice injected intravenously with calf thymus histones (75 mg per kg) with or without APC (5 mg per kg). (b–d) Pathological changes of mouse lung three hours after intravenous injection of histones (50 mg per kg). Immunofluorescence staining for neutrophil elastase detected massive neutrophil accumulation in the alveolar microvasculature (c: histone treated vs. b: control). Alveolar capillaries are almost fully obstructed by cells, as seen by electron microscopy (d: PMN). (e–f) Histones induce strong alterations of the selective permeability of plasma membranes and subsequent intracellular edema, vacuolization (*) of the intracellular organelles (endoplasmic reticulum, Golgi, and mitochondria), both within endothelial (EC) and type I epithelial cells (ep–I). av, alveolae; cav, caveolae; RBC, red blood cells. Magnification bars: b and c: 50 µm; d, 10 µm; e and f, 500 nm.
    Figure Legend Snippet: Intravenous injection of histones elicits inflammatory and cell injury responses. (a) Survival rates of mice injected intravenously with calf thymus histones (75 mg per kg) with or without APC (5 mg per kg). (b–d) Pathological changes of mouse lung three hours after intravenous injection of histones (50 mg per kg). Immunofluorescence staining for neutrophil elastase detected massive neutrophil accumulation in the alveolar microvasculature (c: histone treated vs. b: control). Alveolar capillaries are almost fully obstructed by cells, as seen by electron microscopy (d: PMN). (e–f) Histones induce strong alterations of the selective permeability of plasma membranes and subsequent intracellular edema, vacuolization (*) of the intracellular organelles (endoplasmic reticulum, Golgi, and mitochondria), both within endothelial (EC) and type I epithelial cells (ep–I). av, alveolae; cav, caveolae; RBC, red blood cells. Magnification bars: b and c: 50 µm; d, 10 µm; e and f, 500 nm.

    Techniques Used: Injection, Mouse Assay, Immunofluorescence, Staining, Electron Microscopy, Permeability

    22) Product Images from "Caveolin-Induced Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Increases Arsenite Cytotoxicity"

    Article Title: Caveolin-Induced Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Increases Arsenite Cytotoxicity

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.7.2407-2414.2003

    Differential sensitivity of arsenite-treated HeLa cells expressing different levels of caveolin. (A) HeLa cells transfected with either empty vector pcDNA3.1 (HeLa-V) or pcDNA3.1-caveolin-1 (HeLa-C1) to overexpress caveolin-1 were treated with arsenite, and caveolin-1 and p-Akt expression were subsequently evaluated by Western blot analysis. (B) HeLa-V and HeLa-Cav1 cells were harvested, and Akt was immunoprecipitated. Akt kinase activity was evaluated by using histone H2B as a substrate. (C) Graph depicting the quantitation of clonogenicity assays following exposure to the doses of arsenite shown for 24 h. (D) Photograph illustrating the different numbers of colonies formed by HeLa-V and HeLa-C1 populations after treatment with 10 μM arsenite for 24 h, replating for clonogenicity assay, and staining with crystal violet. Data represent the means and standard deviations from three independent experiments.
    Figure Legend Snippet: Differential sensitivity of arsenite-treated HeLa cells expressing different levels of caveolin. (A) HeLa cells transfected with either empty vector pcDNA3.1 (HeLa-V) or pcDNA3.1-caveolin-1 (HeLa-C1) to overexpress caveolin-1 were treated with arsenite, and caveolin-1 and p-Akt expression were subsequently evaluated by Western blot analysis. (B) HeLa-V and HeLa-Cav1 cells were harvested, and Akt was immunoprecipitated. Akt kinase activity was evaluated by using histone H2B as a substrate. (C) Graph depicting the quantitation of clonogenicity assays following exposure to the doses of arsenite shown for 24 h. (D) Photograph illustrating the different numbers of colonies formed by HeLa-V and HeLa-C1 populations after treatment with 10 μM arsenite for 24 h, replating for clonogenicity assay, and staining with crystal violet. Data represent the means and standard deviations from three independent experiments.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Activity Assay, Quantitation Assay, Staining

    Caveolin-induced changes in Akt and MAP kinase signaling pathways after exposure of 293 cells to arsenite. (A) 293-V, 293-Cav25, and 293-Cav31 cells were treated with 25 μM arsenite for the indicated times, whereupon Western blot analysis was carried out to assess the levels of oligomerized caveolin, monomeric caveolin, p-Akt, Akt, p-GSK-3 α/β, p-ERK, p-JNK, and p-p38 in 50 μg of whole-cell lysate. (B) 293-V and 293-Cav25 cells were harvested, and Akt was immunoprecipitated from lysates with a polyclonal anti-Akt antibody. Akt kinase activity was assessed by using histone H2B as a substrate.
    Figure Legend Snippet: Caveolin-induced changes in Akt and MAP kinase signaling pathways after exposure of 293 cells to arsenite. (A) 293-V, 293-Cav25, and 293-Cav31 cells were treated with 25 μM arsenite for the indicated times, whereupon Western blot analysis was carried out to assess the levels of oligomerized caveolin, monomeric caveolin, p-Akt, Akt, p-GSK-3 α/β, p-ERK, p-JNK, and p-p38 in 50 μg of whole-cell lysate. (B) 293-V and 293-Cav25 cells were harvested, and Akt was immunoprecipitated from lysates with a polyclonal anti-Akt antibody. Akt kinase activity was assessed by using histone H2B as a substrate.

    Techniques Used: Western Blot, Immunoprecipitation, Activity Assay

    23) Product Images from "Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice"

    Article Title: Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice

    Journal: Blood

    doi: 10.1182/blood-2015-05-644401

    aPC inhibits PAR2 activation by the ternary TF-fVIIa-fXa complex. (A) aPC suppresses histone-induced Peli1 expression independent of fV. Quantitative PCR measurement of Peli1 mRNA in RAW cells treated for 3 hours with the indicated reagents (100 ng/mL
    Figure Legend Snippet: aPC inhibits PAR2 activation by the ternary TF-fVIIa-fXa complex. (A) aPC suppresses histone-induced Peli1 expression independent of fV. Quantitative PCR measurement of Peli1 mRNA in RAW cells treated for 3 hours with the indicated reagents (100 ng/mL

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    24) Product Images from "Atorvastatin ameliorates cerebral vasospasm and early brain injury after subarachnoid hemorrhage and inhibits caspase-dependent apoptosis pathway"

    Article Title: Atorvastatin ameliorates cerebral vasospasm and early brain injury after subarachnoid hemorrhage and inhibits caspase-dependent apoptosis pathway

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-10-7

    Cell death assay of DNA-fragmentation after SAH . The results showed that apoptotic-related DNA-fragmentation was increased significantly in SAH rats compared with that in SC rats at 24 hour in basal cortex. Atorvastatin decreased the DNA-fragmentation markedly (*, P
    Figure Legend Snippet: Cell death assay of DNA-fragmentation after SAH . The results showed that apoptotic-related DNA-fragmentation was increased significantly in SAH rats compared with that in SC rats at 24 hour in basal cortex. Atorvastatin decreased the DNA-fragmentation markedly (*, P

    Techniques Used:

    25) Product Images from "Herpes simplex virus protein kinase US3 activates and functionally overlaps protein kinase A to block apoptosis"

    Article Title: Herpes simplex virus protein kinase US3 activates and functionally overlaps protein kinase A to block apoptosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0403160101

    Phosphorylation of known PKA substrates by GST-U S 3 kinase. Two micrograms ( A )or4 μg( B ) of the indicated peptides were reacted with 1 μg of purified GST or GST-Us3 attached to glutathione Sepharose beads, as described in the text. Phosphorylated substrates were adsorbed onto cellulose phosphate paper filters, and radioactivity was measured with a scintillation counter. The data show the increase in cpm over a mock reaction carried out with no substrate. ( C ) Four micrograms of histone H1 were reacted with 1 μg of purified GST or GST-U S 3 attached to glutathione Sepharose beads, as described in the text. Samples were subjected to polyacrylamide gel electrophoresis, nitrocellulose membrane transfer, and autoradiography.
    Figure Legend Snippet: Phosphorylation of known PKA substrates by GST-U S 3 kinase. Two micrograms ( A )or4 μg( B ) of the indicated peptides were reacted with 1 μg of purified GST or GST-Us3 attached to glutathione Sepharose beads, as described in the text. Phosphorylated substrates were adsorbed onto cellulose phosphate paper filters, and radioactivity was measured with a scintillation counter. The data show the increase in cpm over a mock reaction carried out with no substrate. ( C ) Four micrograms of histone H1 were reacted with 1 μg of purified GST or GST-U S 3 attached to glutathione Sepharose beads, as described in the text. Samples were subjected to polyacrylamide gel electrophoresis, nitrocellulose membrane transfer, and autoradiography.

    Techniques Used: Purification, Radioactivity, Polyacrylamide Gel Electrophoresis, Autoradiography

    26) Product Images from "Mutations in Drosophila Greatwall/Scant Reveal Its Roles in Mitosis and Meiosis and Interdependence with Polo Kinase"

    Article Title: Mutations in Drosophila Greatwall/Scant Reveal Its Roles in Mitosis and Meiosis and Interdependence with Polo Kinase

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030200

    Gwl-K97M Is Hyperactive (A) Effects of overexpressing gwl-long-wt or gwl-long - K97M ( Scant ) on female fertility. Mat α -Tub-Gal4 and the UASp-gwl transgenes were present in only one copy on the same chromosome in all flies tested. Single females of the indicated genotypes with 3 WT ( Oregon R) males laid eggs for 3 consecutive d. Numbers are averages of hatched adult progeny (± standard error of the mean) per day for 12 females of each genotype (three females of each of four independent transgenic lines per genotype = 36 samples). The 100% reference comes from the observation of 100% egg hatch and no larval or pupal lethality. (B) Effect of overexpressing gwl-long-wt or - K97M ( Scant ) on embryonic mitosis. Embryos were laid by mothers of the indicated genotypes and treated as in Figure 2 A. Scale bars are in μm. (C) Endogenous Scant protein is not more abundant than Gwl-wt in embryos. Western blots for Gwl and Polo from embryos from mothers of the indicated genotypes. *, cross-reactive band that serves as a loading control. (D) Gwl-K97M is hyperactive in vitro with altered specificity. Myc-tagged forms of Gwl-wt, K87R (kinase dead), and K97M (Scant) were expressed in Dmel stable cell lines. Myc immunoprecipitations were carried out and the kinase activity on Histones H1, H3, Casein, and Myelin Basic Protein was assayed on beads. Note that the Gwl-K97M was always expressed at lower levels in stable cell lines, suggesting toxicity for this protein.
    Figure Legend Snippet: Gwl-K97M Is Hyperactive (A) Effects of overexpressing gwl-long-wt or gwl-long - K97M ( Scant ) on female fertility. Mat α -Tub-Gal4 and the UASp-gwl transgenes were present in only one copy on the same chromosome in all flies tested. Single females of the indicated genotypes with 3 WT ( Oregon R) males laid eggs for 3 consecutive d. Numbers are averages of hatched adult progeny (± standard error of the mean) per day for 12 females of each genotype (three females of each of four independent transgenic lines per genotype = 36 samples). The 100% reference comes from the observation of 100% egg hatch and no larval or pupal lethality. (B) Effect of overexpressing gwl-long-wt or - K97M ( Scant ) on embryonic mitosis. Embryos were laid by mothers of the indicated genotypes and treated as in Figure 2 A. Scale bars are in μm. (C) Endogenous Scant protein is not more abundant than Gwl-wt in embryos. Western blots for Gwl and Polo from embryos from mothers of the indicated genotypes. *, cross-reactive band that serves as a loading control. (D) Gwl-K97M is hyperactive in vitro with altered specificity. Myc-tagged forms of Gwl-wt, K87R (kinase dead), and K97M (Scant) were expressed in Dmel stable cell lines. Myc immunoprecipitations were carried out and the kinase activity on Histones H1, H3, Casein, and Myelin Basic Protein was assayed on beads. Note that the Gwl-K97M was always expressed at lower levels in stable cell lines, suggesting toxicity for this protein.

    Techniques Used: Transgenic Assay, Western Blot, In Vitro, Stable Transfection, Activity Assay

    27) Product Images from "Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo"

    Article Title: Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006429

    Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes. (A and B) Targeting strategy to knock-in Flag and HA tags into the cyclin E1 locus to generate N-terminally tagged cyclin E1 Ntag (A) and C-terminally tagged cyclin E1 Ctag alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [ 8 ]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin E1 +/Ntag , cyclin E1 +/Ctag , and cyclin E1 Ntag/Ctag embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for in vitro kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [ 8 ]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [ 8 ]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [ 8 ].
    Figure Legend Snippet: Generation of tagged cyclin E1 knock-In mice and analyses of cyclin E1-containing protein complexes. (A and B) Targeting strategy to knock-in Flag and HA tags into the cyclin E1 locus to generate N-terminally tagged cyclin E1 Ntag (A) and C-terminally tagged cyclin E1 Ctag alleles (B). The exons are shown as green boxes, Flag tag as a blue box, and HA tag as a red box. Start and stop codons are marked with orange and yellow arrowheads, respectively. The hygromycin resistance cassette (Hyg) with flanking loxP sequences (filled arrows) is also indicated. Restriction enzyme recognition sites: E, EcoRI; A, AflII; Sc, ScaI; N, NotI; X, XhoI; K, KpnI; S, SpeI; P, PmeI; Hp, HpaI. Note that panel (A) was shown in ref [ 8 ]. (C) Western blot analysis of wild-type control (Ctrl), heterozygous cyclin E1 +/Ntag , cyclin E1 +/Ctag , and cyclin E1 Ntag/Ctag embryonic stem cells probed with anti-cyclin E1 and -HA antibodies. Actin served as a loading control. Forth panel: cyclin E1 was immunoprecipitated with anti-Flag antibody and the immunoblots were probed with anti-Cdk2 antibody. Fifth panel: anti-Flag immunoprecipitates were used for in vitro kinase reactions using histone H1 as a substrate. (D) Same analyses as in (C) using spleens of homozygous knock-in mice. Lanes 1–2 in panels (C and D) were previously shown in [ 8 ]. (E) Cyclin E levels detected by western blotting in the indicated organs of 1-month-old mice and in embryonic brain (day E14.5). Actin served as a loading control. The last two lanes (Brain) were previously shown in [ 8 ]. (F) Quantification of cyclin E levels in different organs, normalized against actin (from E). (G) Protein lysates from brains and testes of adult tagged cyclin E1 knock-in mice were separated by gel-filtration chromatography. Fractions containing protein complexes of the indicated molecular weights were analyzed by western blotting for cyclin E using an anti-HA antibody. (H) Cyclin E1-associated proteins were purified from the indicated organs of tagged cyclin E1 knock-in (KI) mice, or from control mice (Ctrl, ‘mock’ purifications) by sequential immunoaffinity purifications with anti-Flag and -HA antibodies, and 10% of the final eluate was resolved on PAGE gels and silver-stained. Arrows indicate bands corresponding to cyclin E1. Panels representing embryonic and adult brains were previously shown in [ 8 ].

    Techniques Used: Knock-In, Mouse Assay, FLAG-tag, Western Blot, Immunoprecipitation, In Vitro, Filtration, Chromatography, Purification, Polyacrylamide Gel Electrophoresis, Staining

    Identification of Mybl1 and Dmrtc2 as cyclin E-Cdk2 phosphorylation substrates in the testes. (A) A diagram illustrating cyclin E1-interactome in testes, consisting of highest-confidence ‘core’ interactors (green nodes), and lower-confidence Category 3 interactors (blue nodes) that were included to the interactome based on their reported interaction with core interactors in the STRING database (see S1 Appendix ). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other ‘core’ interactors. Red arrows indicate proteins that were previously implicated to play important roles in spermatogenesis. (B) Interaction between endogenous Cdk2/cyclin E and Mybl1 and Dmrtc2 in mouse testes, detected by IP–western blotting. Cdk2 or Mybl1 were immunoprecipitated from lysates of testes, and immunoblots were probed with the indicated antibodies. (C) N-terminal fragment (aa 1–201) of Dmrtc2, as well as full length, N-terminal (aa 1–376), and C-terminal (aa 376–752) fragments of Mybl1 were expressed as GST-fusion proteins in E . Coli , purified and subjected to in vitro kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ 32 P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. Red arrowheads point to phosphorylated GST-fusion proteins, orange arrowheads indicate phosphorylated truncated proteins, and blue arrow indicates auto-phosphorylated recombinant cyclin E1-Cdk2. (D) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged substrates (Mybl1 or Dmrtc2). After supplementing cells with 6-Fu-ATPγS, labeling of substrates was evaluated by immunoprecipitating Mybl1 or Dmrtc2 with anti-Flag antibody followed by immunoblotting with an anti-thiophosphate ester antibody. Blue arrowheads indicate ATPγS-labeled Mybl1 and Dmrtc2. The experiment was performed on the same gel as the one shown in Fig 5G , hence the vector control (Vector) is identical. (E) A diagram illustrating amino acid residues in Mybl1 and Dmrtc2, which were phosphorylated by cyclin E-Cdk2.
    Figure Legend Snippet: Identification of Mybl1 and Dmrtc2 as cyclin E-Cdk2 phosphorylation substrates in the testes. (A) A diagram illustrating cyclin E1-interactome in testes, consisting of highest-confidence ‘core’ interactors (green nodes), and lower-confidence Category 3 interactors (blue nodes) that were included to the interactome based on their reported interaction with core interactors in the STRING database (see S1 Appendix ). Solid lines depict STRING-verified interactions. Dashed lines depict an interaction derived from our mass spectrometry analyses between cyclin E1 and a protein that has no known interactions with other ‘core’ interactors. Red arrows indicate proteins that were previously implicated to play important roles in spermatogenesis. (B) Interaction between endogenous Cdk2/cyclin E and Mybl1 and Dmrtc2 in mouse testes, detected by IP–western blotting. Cdk2 or Mybl1 were immunoprecipitated from lysates of testes, and immunoblots were probed with the indicated antibodies. (C) N-terminal fragment (aa 1–201) of Dmrtc2, as well as full length, N-terminal (aa 1–376), and C-terminal (aa 376–752) fragments of Mybl1 were expressed as GST-fusion proteins in E . Coli , purified and subjected to in vitro kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ 32 P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. Red arrowheads point to phosphorylated GST-fusion proteins, orange arrowheads indicate phosphorylated truncated proteins, and blue arrow indicates auto-phosphorylated recombinant cyclin E1-Cdk2. (D) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged substrates (Mybl1 or Dmrtc2). After supplementing cells with 6-Fu-ATPγS, labeling of substrates was evaluated by immunoprecipitating Mybl1 or Dmrtc2 with anti-Flag antibody followed by immunoblotting with an anti-thiophosphate ester antibody. Blue arrowheads indicate ATPγS-labeled Mybl1 and Dmrtc2. The experiment was performed on the same gel as the one shown in Fig 5G , hence the vector control (Vector) is identical. (E) A diagram illustrating amino acid residues in Mybl1 and Dmrtc2, which were phosphorylated by cyclin E-Cdk2.

    Techniques Used: Derivative Assay, Mass Spectrometry, Western Blot, Immunoprecipitation, Purification, In Vitro, Recombinant, Positive Control, Negative Control, Transfection, Labeling, Plasmid Preparation

    Interaction of cyclin E1 with the DREAM complex. (A) A diagram showing in which organ a particular component of the DREAM complex was identified as a cyclin E1-interacting protein by our mass spectrometric analyses. The number of organs in which a given protein was found to associate with cyclin E1 is depicted by the color. (B) Association of cyclin E1 with components of the DREAM complex in the spleens of tagged knock-in mice (KI) was verified by immunoprecipitating (IP) cyclin E1 with anti-Flag antibody followed by immunoblotting with the indicated antibodies. (C) IP followed by re-IP-immunoblotting to demonstrate that cyclin E1, Cdk2 and the DREAM complex components are present within the same multi-protein complex. Cyclin E1 was immunoprecipitated from spleens of KI mice using anti-Flag antibody, protein complexes were eluted with Flag peptides, re-immunoprecipitated with IgG (control) or with anti-p130 antibody, and then immunoblotted with the indicated antibodies. (D) T98G cells were serum starved for 72 hrs (0% FBS). Subsequently, cells were stimulated to re-enter the cell cycle by addition of 20% FBS supplemented with either 0.2% DMSO (control, left two panels) or 20 μM CVT-313 (right two panels), and harvested at the indicated time-points. Cell extracts (whole) as well as anti-Lin37 immunoprecipitates were resolved on 4–15% gradient SDS-PAGE gels and probed with indicated antibodies. Gapdh serves as a loading control. (E) Cyclin E1-Cdk2 kinase can phosphorylate purified recombinant Lin proteins in vitro. Lin9, Lin52 and Lin54 were expressed as GST-fusion proteins in E . Coli , purified and subjected to in vitro kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ 32 P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. (F) A diagram illustrating amino acid residues in human Lin proteins that were phosphorylated by cyclin E-Cdk2. (G) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged Lin37 or vector control. After supplementing cells with 6-Fu-ATPγS, labeling of Lin37 was evaluated by immunoprecipitating Lin37 with an anti-Flag antibody followed by immunoblotting with anti-thiophosphate ester antibody. A blue arrowhead indicates 6-Fu-ATPγS-labeled Lin37.
    Figure Legend Snippet: Interaction of cyclin E1 with the DREAM complex. (A) A diagram showing in which organ a particular component of the DREAM complex was identified as a cyclin E1-interacting protein by our mass spectrometric analyses. The number of organs in which a given protein was found to associate with cyclin E1 is depicted by the color. (B) Association of cyclin E1 with components of the DREAM complex in the spleens of tagged knock-in mice (KI) was verified by immunoprecipitating (IP) cyclin E1 with anti-Flag antibody followed by immunoblotting with the indicated antibodies. (C) IP followed by re-IP-immunoblotting to demonstrate that cyclin E1, Cdk2 and the DREAM complex components are present within the same multi-protein complex. Cyclin E1 was immunoprecipitated from spleens of KI mice using anti-Flag antibody, protein complexes were eluted with Flag peptides, re-immunoprecipitated with IgG (control) or with anti-p130 antibody, and then immunoblotted with the indicated antibodies. (D) T98G cells were serum starved for 72 hrs (0% FBS). Subsequently, cells were stimulated to re-enter the cell cycle by addition of 20% FBS supplemented with either 0.2% DMSO (control, left two panels) or 20 μM CVT-313 (right two panels), and harvested at the indicated time-points. Cell extracts (whole) as well as anti-Lin37 immunoprecipitates were resolved on 4–15% gradient SDS-PAGE gels and probed with indicated antibodies. Gapdh serves as a loading control. (E) Cyclin E1-Cdk2 kinase can phosphorylate purified recombinant Lin proteins in vitro. Lin9, Lin52 and Lin54 were expressed as GST-fusion proteins in E . Coli , purified and subjected to in vitro kinase reactions with the recombinant cyclin E1-Cdk2 in the presence of [γ 32 P]ATP. Recombinant histone H1 was used as a positive control and GST as a negative control. (F) A diagram illustrating amino acid residues in human Lin proteins that were phosphorylated by cyclin E-Cdk2. (G) Wild-type Cdk2 (K2WT) or analog-sensitive Cdk2 (K2AS) were transfected into 293T cells together with cyclin E1 and Flag-tagged Lin37 or vector control. After supplementing cells with 6-Fu-ATPγS, labeling of Lin37 was evaluated by immunoprecipitating Lin37 with an anti-Flag antibody followed by immunoblotting with anti-thiophosphate ester antibody. A blue arrowhead indicates 6-Fu-ATPγS-labeled Lin37.

    Techniques Used: Knock-In, Mouse Assay, Immunoprecipitation, SDS Page, Purification, Recombinant, In Vitro, Positive Control, Negative Control, Transfection, Plasmid Preparation, Labeling

    Quantitative proteomic (iTRAQ) analysis of cyclin E1-interacting proteins in mouse organs in the absence of Cdk2. (A) Relative abundance of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of Cdk2 -/- /cyclin E1 Ntag/Ntag mice, as compared to Cdk2 +/+ /cyclin E1 Ntag/Ntag animals, was determined by iTRAQ labeling and LC-MS. (B) The amount of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of wild-type (Ctrl), Cdk2 +/+ /cyclin E1 Ntag/Ntag (KI), and Cdk2 -/- /cyclin E1 Ntag/Ntag (Cdk2 -/- ) mice was gauged by immunoprecipitation with an anti-Flag antibody and immunoblotting with the indicated antibodies. Abundance of each protein in total lysates (whole) is also shown. (C) Spleens from wild-type mice were incubated with 20 μM CVT-313 (+) or with vehicle only (-). Association of cyclin E1 with Cdk2, Cdk1, Cdk4 and Cdk5 was assessed by IP–western blotting. Whole, whole cell lysate from vehicle only-treated mice. Lower panel: To ensure that CVT-313 treatment inhibited Cdk2 kinase activity, Cdk2 was immunoprecipitated from lysates and used for in vitro kinase reactions with histone H1 as a substrate. Note that CVT-313 treatment strongly decreased Cdk2 kinase activity.
    Figure Legend Snippet: Quantitative proteomic (iTRAQ) analysis of cyclin E1-interacting proteins in mouse organs in the absence of Cdk2. (A) Relative abundance of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of Cdk2 -/- /cyclin E1 Ntag/Ntag mice, as compared to Cdk2 +/+ /cyclin E1 Ntag/Ntag animals, was determined by iTRAQ labeling and LC-MS. (B) The amount of cyclin E1-associated Cdk1, Cdk2, Cdk4, Cdk5 and p107 in the spleens of wild-type (Ctrl), Cdk2 +/+ /cyclin E1 Ntag/Ntag (KI), and Cdk2 -/- /cyclin E1 Ntag/Ntag (Cdk2 -/- ) mice was gauged by immunoprecipitation with an anti-Flag antibody and immunoblotting with the indicated antibodies. Abundance of each protein in total lysates (whole) is also shown. (C) Spleens from wild-type mice were incubated with 20 μM CVT-313 (+) or with vehicle only (-). Association of cyclin E1 with Cdk2, Cdk1, Cdk4 and Cdk5 was assessed by IP–western blotting. Whole, whole cell lysate from vehicle only-treated mice. Lower panel: To ensure that CVT-313 treatment inhibited Cdk2 kinase activity, Cdk2 was immunoprecipitated from lysates and used for in vitro kinase reactions with histone H1 as a substrate. Note that CVT-313 treatment strongly decreased Cdk2 kinase activity.

    Techniques Used: Mouse Assay, Labeling, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Incubation, Western Blot, Activity Assay, In Vitro

    28) Product Images from "HUA ENHANCER3 reveals a role for a cyclin-dependent protein kinase in the specification of floral organ identity in Arabidopsis"

    Article Title: HUA ENHANCER3 reveals a role for a cyclin-dependent protein kinase in the specification of floral organ identity in Arabidopsis

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.01187

    HEN3 has CTD kinase activity. HEN3-HA transgenic plants or control non-transgenic plants were immunoprecipitated with an anti-HA monoclonal antibody, and the immunoprecipitate was used in a kinase assay with 10 µg purified histone H1, 6×His-CTD
    Figure Legend Snippet: HEN3 has CTD kinase activity. HEN3-HA transgenic plants or control non-transgenic plants were immunoprecipitated with an anti-HA monoclonal antibody, and the immunoprecipitate was used in a kinase assay with 10 µg purified histone H1, 6×His-CTD

    Techniques Used: Activity Assay, Transgenic Assay, Immunoprecipitation, Kinase Assay, Purification

    29) Product Images from "Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors"

    Article Title: Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors

    Journal: Yonsei Medical Journal

    doi: 10.3349/ymj.2018.59.8.995

    A nonradioisotopic assay method to measure Cdk1 kinase activity. We used two kinds of Cdk1 kinases, one was Cdk1 IP and the other was commercially available Cdk1. The Cdk1 IP was prepared from HeLa cell extracts treated with 250 nM nocodazole, and the commercial Cdk1 kinase was purchased. These two kinds of Cdk1 were used to measure Cdk1 kinase activity in the presence (lanes 2, 6) or absence (lanes 1, 3, 4, 5, 7, 8) of 1 mM olomoucine (a Cdk1 kinase inhibitor) using Western blotting with 1 μg of Rb (A) or 2 μg of histone H1 (B) as a substrate. The Cdk1 kinase activity was determined by phosphorylation levels of each substrate using the anti-pSer 795 Rb antibody or anti-pThr/Pro motif-recognizing antibody, respectively. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.
    Figure Legend Snippet: A nonradioisotopic assay method to measure Cdk1 kinase activity. We used two kinds of Cdk1 kinases, one was Cdk1 IP and the other was commercially available Cdk1. The Cdk1 IP was prepared from HeLa cell extracts treated with 250 nM nocodazole, and the commercial Cdk1 kinase was purchased. These two kinds of Cdk1 were used to measure Cdk1 kinase activity in the presence (lanes 2, 6) or absence (lanes 1, 3, 4, 5, 7, 8) of 1 mM olomoucine (a Cdk1 kinase inhibitor) using Western blotting with 1 μg of Rb (A) or 2 μg of histone H1 (B) as a substrate. The Cdk1 kinase activity was determined by phosphorylation levels of each substrate using the anti-pSer 795 Rb antibody or anti-pThr/Pro motif-recognizing antibody, respectively. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.

    Techniques Used: Activity Assay, Western Blot

    Optimization of several conditions for the Cdk1 kinase assay. Several key factors were examined to optimize the conditions of the Cdk1 kinase assay. The basic procedure of the reaction was described in the Materials and Methods section with the exception of the indicated protein amounts from HeLa cell extracts to obtain Cdk1 IP, substrate amounts, ATP concentrations, and reaction times using Rb or histone H1 as a substrate. All examined parameters were positively correlated with Cdk1 kinase activity, except for the excess concentration of ATP. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.
    Figure Legend Snippet: Optimization of several conditions for the Cdk1 kinase assay. Several key factors were examined to optimize the conditions of the Cdk1 kinase assay. The basic procedure of the reaction was described in the Materials and Methods section with the exception of the indicated protein amounts from HeLa cell extracts to obtain Cdk1 IP, substrate amounts, ATP concentrations, and reaction times using Rb or histone H1 as a substrate. All examined parameters were positively correlated with Cdk1 kinase activity, except for the excess concentration of ATP. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.

    Techniques Used: Kinase Assay, Activity Assay, Concentration Assay

    30) Product Images from "Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress"

    Article Title: Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress

    Journal: Oncotarget

    doi: 10.18632/oncotarget.22360

    Nrf2 activation is required for MIND4-17-mediated cytoprotection OB-6 cells, stably expressing lentiviral scramble non-sense control shRNA (“sh-scr”) or Nrf2 shRNA (“sh-Nrf2, sequence -1/−2”) ( A - E ), as well as lentiviral Keap1 shRNA (“sh-Keap1”) ( F - J ), were treated with MIND4-17 (3 μM) for 3 hours, expressions of listed proteins were presented (A and F); mRNA expressions of listed genes were also shown (B, C, G and H); Cells were further treated with/out hydrogen peroxide (“H 2 O 2 ”, 200 μM) for indicated time period, cell viability (CCK-8 assay, D and I) and apoptosis (Histone DNA ELISA assay, E and J) were also tested. For H 2 O 2 experiments, MIND4-17 (3 μM) were pretreated before H 2 O 2 for 1 hour. For all the assays, the exactsame number of viable cells with the applied shRNA was initially plated into each well. Data were presented as mean (n=5) ± standard deviation (SD). # p
    Figure Legend Snippet: Nrf2 activation is required for MIND4-17-mediated cytoprotection OB-6 cells, stably expressing lentiviral scramble non-sense control shRNA (“sh-scr”) or Nrf2 shRNA (“sh-Nrf2, sequence -1/−2”) ( A - E ), as well as lentiviral Keap1 shRNA (“sh-Keap1”) ( F - J ), were treated with MIND4-17 (3 μM) for 3 hours, expressions of listed proteins were presented (A and F); mRNA expressions of listed genes were also shown (B, C, G and H); Cells were further treated with/out hydrogen peroxide (“H 2 O 2 ”, 200 μM) for indicated time period, cell viability (CCK-8 assay, D and I) and apoptosis (Histone DNA ELISA assay, E and J) were also tested. For H 2 O 2 experiments, MIND4-17 (3 μM) were pretreated before H 2 O 2 for 1 hour. For all the assays, the exactsame number of viable cells with the applied shRNA was initially plated into each well. Data were presented as mean (n=5) ± standard deviation (SD). # p

    Techniques Used: Activation Assay, Stable Transfection, Expressing, shRNA, Sequencing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    31) Product Images from "Preclinical study of cinobufagin as a promising anti-colorectal cancer agent"

    Article Title: Preclinical study of cinobufagin as a promising anti-colorectal cancer agent

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13519

    Cinobufagin (CBG) provokes apoptosis in CRC cells Listed cancer cells or non-cancerous epithelial cells were treated with/out designated concentrations of cinobufagin (CBG, 1-250 ng/mL), cells were further cultured for indicated time; Cell apoptosis was tested by TUNEL staining assay A , B , G and H. , histone-DNA ELISA assay C and D. and Annexin V FACS assay ( E and F. , quantified results were presented at the right panels). Experiments in this figure were repeated three times, with similar results obtained. * p
    Figure Legend Snippet: Cinobufagin (CBG) provokes apoptosis in CRC cells Listed cancer cells or non-cancerous epithelial cells were treated with/out designated concentrations of cinobufagin (CBG, 1-250 ng/mL), cells were further cultured for indicated time; Cell apoptosis was tested by TUNEL staining assay A , B , G and H. , histone-DNA ELISA assay C and D. and Annexin V FACS assay ( E and F. , quantified results were presented at the right panels). Experiments in this figure were repeated three times, with similar results obtained. * p

    Techniques Used: Cell Culture, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, FACS

    32) Product Images from "Systems analysis reveals a transcriptional reversal of the mesenchymal phenotype induced by SNAIL-inhibitor GN-25"

    Article Title: Systems analysis reveals a transcriptional reversal of the mesenchymal phenotype induced by SNAIL-inhibitor GN-25

    Journal: BMC Systems Biology

    doi: 10.1186/1752-0509-7-85

    GN-25 induces apoptosis in HMLE cells. HMLE cells were exposed to different concentrations of GN-25 as indicated and incubated for 72 hrs. Apoptosis was evaluated using Annexin V FITC assay (A) and Histone DNA ELISA assays (B) . Values are representative of three independent experiments. (C) Cells were incubated with either scrambled siRNA or SNAI2 siRNA (Santa Cruz Biotechnology) for 24 hrs according to previously published methods [26], siRNA silenced cells were seeded at a density of 50,000 cells per well in six well plate and further exposed to GN-25 (20 μM) for additional 72 hrs. Apoptosis was evaluated using Histone DNA ELISA assay. Graphs are representative of three independent experiments. ** represents statistically significant p
    Figure Legend Snippet: GN-25 induces apoptosis in HMLE cells. HMLE cells were exposed to different concentrations of GN-25 as indicated and incubated for 72 hrs. Apoptosis was evaluated using Annexin V FITC assay (A) and Histone DNA ELISA assays (B) . Values are representative of three independent experiments. (C) Cells were incubated with either scrambled siRNA or SNAI2 siRNA (Santa Cruz Biotechnology) for 24 hrs according to previously published methods [26], siRNA silenced cells were seeded at a density of 50,000 cells per well in six well plate and further exposed to GN-25 (20 μM) for additional 72 hrs. Apoptosis was evaluated using Histone DNA ELISA assay. Graphs are representative of three independent experiments. ** represents statistically significant p

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

    33) Product Images from "Monomethyl Histone H3 Lysine 4 as an Epigenetic Mark for Silenced Euchromatin in Chlamydomonas W⃞"

    Article Title: Monomethyl Histone H3 Lysine 4 as an Epigenetic Mark for Silenced Euchromatin in Chlamydomonas W⃞

    Journal: The Plant Cell

    doi: 10.1105/tpc.105.034165

    The Mut11-TAPp Complex(es) Methylates Histones H3, H2A, and H4, and Set1p Is an H3K4 HMTase.
    Figure Legend Snippet: The Mut11-TAPp Complex(es) Methylates Histones H3, H2A, and H4, and Set1p Is an H3K4 HMTase.

    Techniques Used:

    34) Product Images from "Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul"

    Article Title: Targeting beta-Catenin signaling to induce apoptosis in human breast cancer cells by z-Guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/1472-6882-13-203

    Immunoblotting for β-Catenin using lysates from MCF-7 (A) or MDA-MB-231 (C) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA–transfected cells. Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (B) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P
    Figure Legend Snippet: Immunoblotting for β-Catenin using lysates from MCF-7 (A) or MDA-MB-231 (C) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA–transfected cells. Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (B) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or β-Catenin -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P

    Techniques Used: Multiple Displacement Amplification, Transfection

    GL induced apoptosis in MCF-7 and MDA-MB-231 cells, but not in HMEC, determined by (A and D) quantitation of cytoplasmic histone associated DNA fragmentation, and (B) flow cytomitry analysis of Caspase 3 activity. Cells were treated with the indicated concentrations of GL or z-Gug (C) or DMSO (control) for 24 hours. Results are expressed as enrichment factor relative to cells treated with DMSO (control). Results are mean±SE ( n =3). *Significantly different ( P
    Figure Legend Snippet: GL induced apoptosis in MCF-7 and MDA-MB-231 cells, but not in HMEC, determined by (A and D) quantitation of cytoplasmic histone associated DNA fragmentation, and (B) flow cytomitry analysis of Caspase 3 activity. Cells were treated with the indicated concentrations of GL or z-Gug (C) or DMSO (control) for 24 hours. Results are expressed as enrichment factor relative to cells treated with DMSO (control). Results are mean±SE ( n =3). *Significantly different ( P

    Techniques Used: Multiple Displacement Amplification, Quantitation Assay, Flow Cytometry, Activity Assay

    Immunoblotting for TCF-4 using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 μmol/L GL for 24 h. Immunoblotting for TCF-4 using lysates from MCF-7 (C) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or TCF-4 -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated control cells (A-B) or relative to DMSO-treated nonspecific control siRNA–transfected cells (C-D) . Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (E) or MDA-MB-231 (F) cells transiently transfected with a control nonspecific siRNA or TCF-4-targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P
    Figure Legend Snippet: Immunoblotting for TCF-4 using lysates from MCF-7 (A) or MDA-MB-231 (B) cells treated with DMSO (control) or 5 μmol/L GL for 24 h. Immunoblotting for TCF-4 using lysates from MCF-7 (C) or MDA-MB-231 (D) cells transiently transfected with a control nonspecific siRNA or TCF-4 -targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated control cells (A-B) or relative to DMSO-treated nonspecific control siRNA–transfected cells (C-D) . Cytoplasmic histone-associated apoptotic DNA fragmentation in MCF-7 (E) or MDA-MB-231 (F) cells transiently transfected with a control nonspecific siRNA or TCF-4-targeted siRNA and treated for 24 h with DMSO or 5 μmol/L GL. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean ( n =3); bars, SE. *, P

    Techniques Used: Multiple Displacement Amplification, Transfection

    35) Product Images from "CAK-independent Activation of CDK6 by a Viral Cyclin"

    Article Title: CAK-independent Activation of CDK6 by a Viral Cyclin

    Journal: Molecular Biology of the Cell

    doi:

    Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.
    Figure Legend Snippet: Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.

    Techniques Used: Activity Assay, Transfection, Expressing, Immunoprecipitation, Autoradiography, SDS Page

    Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.
    Figure Legend Snippet: Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.

    Techniques Used: Inhibition, Activity Assay, Mutagenesis, Purification, Infection

    Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.
    Figure Legend Snippet: Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.
    Figure Legend Snippet: CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    36) Product Images from "CAK-independent Activation of CDK6 by a Viral Cyclin"

    Article Title: CAK-independent Activation of CDK6 by a Viral Cyclin

    Journal: Molecular Biology of the Cell

    doi:

    Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.
    Figure Legend Snippet: Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.

    Techniques Used: Activity Assay, Transfection, Expressing, Immunoprecipitation, Autoradiography, SDS Page

    Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.
    Figure Legend Snippet: Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.

    Techniques Used: Inhibition, Activity Assay, Mutagenesis, Purification, Infection

    Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.
    Figure Legend Snippet: Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.
    Figure Legend Snippet: CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    37) Product Images from "CAK-independent Activation of CDK6 by a Viral Cyclin"

    Article Title: CAK-independent Activation of CDK6 by a Viral Cyclin

    Journal: Molecular Biology of the Cell

    doi:

    Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.
    Figure Legend Snippet: Activity of CDK6/KSHV-cyclin complexes in cells. U2OS cells transfected with Myc-KSHV-cyclin, cyclin D1, HA-CDK6, HA-CDK6 T177A , and p16 expression vectors as indicated were lysed at 48 h post-transfection, and CDK6 was immunoprecipitated via its HA-tag. Kinase activities toward GST-Rb and histone H1 were determined as detailed in MATERIALS AND METHODS. Phosphorylated substrates were detected by autoradiography following 10% SDS-PAGE. Bottom, immunoblot of samples with antibody against CDK6. The upper band represents the transfected CDK6-HA and the lower band represents the endogenous CDK6.

    Techniques Used: Activity Assay, Transfection, Expressing, Immunoprecipitation, Autoradiography, SDS Page

    Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.
    Figure Legend Snippet: Phosphorylation-dependent sensitivity of CDK6 to inhibition by p16. (A) GST-CDK6/cyclin D1 complexes were treated with decreasing amounts of p16 (left) or p27 (right) and then assayed for their Rb kinase activity. (B) CDK6/KSHV-cyclin complexes were treated with decreasing amounts of p16 or p27 and assayed for their Rb kinase (top), histone H1 kinase (middle), and CTD kinase (bottom) activities. (C) Same experiment as B except that CDK6 was first phosphorylated on Thr-177 by Cak1p. (D) Same experiment as B using mutant GST-CDK6 T177A /KSHV–cyclin complexes (that cannot be phosphorylated by CAK). In A–D, lanes 1 contained no inhibitors. Mass ratios of inhibitor: CDK6 were 0 (lanes 1), 10:1 (lanes 2), 5:1 (lanes 3), 2.5:1 (lanes 4), 1.2:1 (lanes 5), 0.3:1 (lanes 6), 0.09:1 (lanes 7), and 0.03:1 (lanes 8). Note that GST-CDK6/cyclin D1 complexes were purified from insect cells after coinfection with baculoviruses. Monomeric GST-CDK6 was obtained from singly infected insect cells and GST-CDK6 T177A was purified from bacteria. Asterisk represents phosphorylation of the KSHV-cyclin.

    Techniques Used: Inhibition, Activity Assay, Mutagenesis, Purification, Infection

    Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.
    Figure Legend Snippet: Activation of CDK2 by KSHV-cyclin is CAK dependent. CDK2 was incubated with increasing amounts of purified KSHV-cyclin and assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK2 were used: CDK2 phosphorylated on Thr-160 by Cak1p (●), CDK2 (○), and GST-CDK2 T160A (□). First data point contained no KSHV-cyclin. Mass ratios of KSHV-cyclin: CDK2 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. CDK2 was purified from insect cells after infection with a baculovirus; GST-CDK2 T160A and KSHV-cyclin were purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.
    Figure Legend Snippet: CAK-independent activation of CDK6 by KSHV-cyclin. GST-CDK6 was incubated with increasing amounts of purified KSHV-cyclin and then assayed for Rb kinase (A), histone H1 kinase (B), and CTD kinase (C) activities. The following forms of CDK6 were used: GST-CDK6 (○), GST-CDK6 phosphorylated on Thr-177 by Cak1p (●), and GST-CDK6 T177A (□). Mass ratios of KSHV-cyclin: CDK6 were 0, 1:10, 1:2.5, 1:1.4, 1:1, 4:1, 7:1, and 10:1. Data were fit to the Michealis-Menten equation and the corresponding velocities are shown in D. Note that wild-type GST-CDK6 was purified after baculoviral infection from insect cells, whereas GST-CDK6 T177A was purified from bacteria.

    Techniques Used: Activation Assay, Incubation, Purification, Infection

    38) Product Images from "Extracellular histones are essential effectors of C5aR- and C5L2-mediated tissue damage and inflammation in acute lung injury"

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

    Journal: The FASEB Journal

    doi: 10.1096/fj.13-236380

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

    Techniques Used: Purification, Chromogenic Assay, Staining

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

    Techniques Used: Purification

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

    Techniques Used: Neutralization, Permeability

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

    Techniques Used: Mouse Assay, Western Blot, Negative Control

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

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

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

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

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

    Techniques Used: Purification

    39) Product Images from "Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors"

    Article Title: Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors

    Journal: Yonsei Medical Journal

    doi: 10.3349/ymj.2018.59.8.995

    A nonradioisotopic assay method to measure Cdk1 kinase activity. We used two kinds of Cdk1 kinases, one was Cdk1 IP and the other was commercially available Cdk1. The Cdk1 IP was prepared from HeLa cell extracts treated with 250 nM nocodazole, and the commercial Cdk1 kinase was purchased. These two kinds of Cdk1 were used to measure Cdk1 kinase activity in the presence (lanes 2, 6) or absence (lanes 1, 3, 4, 5, 7, 8) of 1 mM olomoucine (a Cdk1 kinase inhibitor) using Western blotting with 1 μg of Rb (A) or 2 μg of histone H1 (B) as a substrate. The Cdk1 kinase activity was determined by phosphorylation levels of each substrate using the anti-pSer 795 Rb antibody or anti-pThr/Pro motif-recognizing antibody, respectively. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.
    Figure Legend Snippet: A nonradioisotopic assay method to measure Cdk1 kinase activity. We used two kinds of Cdk1 kinases, one was Cdk1 IP and the other was commercially available Cdk1. The Cdk1 IP was prepared from HeLa cell extracts treated with 250 nM nocodazole, and the commercial Cdk1 kinase was purchased. These two kinds of Cdk1 were used to measure Cdk1 kinase activity in the presence (lanes 2, 6) or absence (lanes 1, 3, 4, 5, 7, 8) of 1 mM olomoucine (a Cdk1 kinase inhibitor) using Western blotting with 1 μg of Rb (A) or 2 μg of histone H1 (B) as a substrate. The Cdk1 kinase activity was determined by phosphorylation levels of each substrate using the anti-pSer 795 Rb antibody or anti-pThr/Pro motif-recognizing antibody, respectively. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.

    Techniques Used: Activity Assay, Western Blot

    Optimization of several conditions for the Cdk1 kinase assay. Several key factors were examined to optimize the conditions of the Cdk1 kinase assay. The basic procedure of the reaction was described in the Materials and Methods section with the exception of the indicated protein amounts from HeLa cell extracts to obtain Cdk1 IP, substrate amounts, ATP concentrations, and reaction times using Rb or histone H1 as a substrate. All examined parameters were positively correlated with Cdk1 kinase activity, except for the excess concentration of ATP. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.
    Figure Legend Snippet: Optimization of several conditions for the Cdk1 kinase assay. Several key factors were examined to optimize the conditions of the Cdk1 kinase assay. The basic procedure of the reaction was described in the Materials and Methods section with the exception of the indicated protein amounts from HeLa cell extracts to obtain Cdk1 IP, substrate amounts, ATP concentrations, and reaction times using Rb or histone H1 as a substrate. All examined parameters were positively correlated with Cdk1 kinase activity, except for the excess concentration of ATP. Cdk1, cyclin-dependent kinase 1; Rb, retinoblastoma protein; IP, immunoprecipitates.

    Techniques Used: Kinase Assay, Activity Assay, Concentration Assay

    40) Product Images from "The Interaction of Herpes Simplex Virus 1 Regulatory Protein ICP22 with the cdc25C Phosphatase Is Enabled In Vitro by Viral Protein Kinases US3 and UL13 "

    Article Title: The Interaction of Herpes Simplex Virus 1 Regulatory Protein ICP22 with the cdc25C Phosphatase Is Enabled In Vitro by Viral Protein Kinases US3 and UL13

    Journal: Journal of Virology

    doi: 10.1128/JVI.02022-07

    Endogenous cdc25C phosphatase activity decreases after HSV-1(F) infection. We examined the phosphatase activity of endogenous cdc25C using a two-step assay. cdc25C was immunoprecipitated from lysates of HEp-2 cells harvested 18 h after infection with HSV-1(F) or mock infection. As a positive control, cdc25C was also immunoprecipitated from M-phase lysates prepared from HEp-2 cells treated for 18 h with nocodazole. Simultaneously, cyclin B1-cdc2 complex was immunoprecipitated from S-phase lysates prepared from HEp-2 cells that were treated for 18 h with hydroxyurea. The immunoprecipitated (I.P.) complexes were rinsed, and each cdc25C complex was mixed with cyclin B1-cdc2 complex from S-phase cells in phosphatase reaction buffer for 15 min at 30°C—the first step of this two-step assay. After the phosphatase reaction, the supernatant was removed and the combined complex beads were incubated with histone H1 kinase buffer containing [γ- 32 P]ATP for 15 min at 30°C—the second step of this two-step assay. Pr oteins were electrophoretically separated, transferred, and analyzed by autoradiography (A). The blots were stained with Ponceau S to detect total protein (B). unsync, unsynchronized.
    Figure Legend Snippet: Endogenous cdc25C phosphatase activity decreases after HSV-1(F) infection. We examined the phosphatase activity of endogenous cdc25C using a two-step assay. cdc25C was immunoprecipitated from lysates of HEp-2 cells harvested 18 h after infection with HSV-1(F) or mock infection. As a positive control, cdc25C was also immunoprecipitated from M-phase lysates prepared from HEp-2 cells treated for 18 h with nocodazole. Simultaneously, cyclin B1-cdc2 complex was immunoprecipitated from S-phase lysates prepared from HEp-2 cells that were treated for 18 h with hydroxyurea. The immunoprecipitated (I.P.) complexes were rinsed, and each cdc25C complex was mixed with cyclin B1-cdc2 complex from S-phase cells in phosphatase reaction buffer for 15 min at 30°C—the first step of this two-step assay. After the phosphatase reaction, the supernatant was removed and the combined complex beads were incubated with histone H1 kinase buffer containing [γ- 32 P]ATP for 15 min at 30°C—the second step of this two-step assay. Pr oteins were electrophoretically separated, transferred, and analyzed by autoradiography (A). The blots were stained with Ponceau S to detect total protein (B). unsync, unsynchronized.

    Techniques Used: Activity Assay, Infection, Immunoprecipitation, Positive Control, Incubation, Autoradiography, Staining

    Related Articles

    Apoptosis Assay:

    Article Title: Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models
    Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA) apoptosis assay Histone associated DNA fragmentation during apoptosis was analyzed using an ELISA kit (Roche Applied Science, Indianapolis, IN, USA). ..

    Purification:

    Article Title: Bovine Herpesvirus 1 Tegument Protein VP22 Interacts with Histones, and the Carboxyl Terminus of VP22 Is Required for Nuclear Localization
    Article Snippet: .. To test our hypothesis that the cellular components that associate with VP22 are histones, purified histones (Roche Molecular Biochemicals, Indianapolis, Ind.) were included in the same overlay assays. .. Figure A reveals that VP22 binds to the purified histones with a pattern similar to that of the total cell lysates, confirming that VP22 associated with histones in vitro.

    Article Title: The Yeast Trimeric Guanine Nucleotide-Binding Protein ? Subunit, Gpa2p, Controls the Meiosis-Specific Kinase Ime2p Activity in Response to Nutrients
    Article Snippet: .. GST and GST-Ime2p purified from E. coli and Ime2p-His6 purified from yeast were assayed in 30 μl of reaction buffer containing 5 μg of histone H1 (Roche Diagnostics) and 5 μCi of [γ-32 P]ATP (6,000 Ci/mmol) with 5 μg of either bovine serum albumin (BSA), Gpa2p-His6 bound to GDP, or Gpa2p-His6 bound to GTPγS. .. PKA (Promega, Heidelberg, Germany) was used as a specificity control.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models
    Article Snippet: .. Enzyme-linked immunosorbent assay (ELISA) apoptosis assay Histone associated DNA fragmentation during apoptosis was analyzed using an ELISA kit (Roche Applied Science, Indianapolis, IN, USA). ..

    Article Title: Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells
    Article Snippet: .. Human lung epithelial apoptosis was quantified 4 and 12 h after treatment with leukocyte elastase, PAR-1 AP, control peptide, and thrombin using the Cell Death Detection ELISA kit (Roche, Mannheim, Germany) that specifically detects the histone region (H1, H2A, H2B, H3, and H4) of mono- and oligonucleosomes that are released during apoptosis. .. Absorbance at 405 nm in a 96-well plate was measured using a microplate reader (THERMO max; Molecular Devices, Sunnyvale, CA).

    Incubation:

    Article Title: Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms
    Article Snippet: .. For the histone H1 and GST-Rb kinase assays, the immunoprecipitates were incubated with kinase assay buffer containing 60 μM cold ATP, 5 μCi of [32 P]ATP, and 5 μg of histone H1 (Roche Molecular Biochemicals) or 1 μg of GST-Rb (Santa Cruz Biochemicals) in a final volume of 30 μl at 37°C for 30 min. .. The products of the reactions were then analyzed on either a 13% (histone H1) or 10% (GST-Rb) SDS-PAGE gel.

    Kinase Assay:

    Article Title: Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms
    Article Snippet: .. For the histone H1 and GST-Rb kinase assays, the immunoprecipitates were incubated with kinase assay buffer containing 60 μM cold ATP, 5 μCi of [32 P]ATP, and 5 μg of histone H1 (Roche Molecular Biochemicals) or 1 μg of GST-Rb (Santa Cruz Biochemicals) in a final volume of 30 μl at 37°C for 30 min. .. The products of the reactions were then analyzed on either a 13% (histone H1) or 10% (GST-Rb) SDS-PAGE gel.

    Recombinant:

    Article Title: Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization
    Article Snippet: .. Histone H2A ubiquitination assays were conducted at room temperature for 1 hour with histone H2A (2.5 μg; Roche Biotech), ubiquitin-activating enzyme E1 (50 μg/mL, BioMol), recombinant human Rad6B (85 μg/mL), ubiquitin (1.25 mg/mL; Roche), 2 mmol/L MgCl2 , 4 mmol/L ATP, and energy regeneration system (Boston Biochem) in reaction buffer (50 mmol/L Tris-HCl, pH 7.5; ). .. To assess TZ compound effects on Rad6B-mediated ubiquitination, reactions containing Rad6B were preincubated with the compounds (25 nmol/L) or vehicle for 1 hour before adding ubiquitin and histone H2A.

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    Roche enzyme linked immunosorbent assay elisa apoptosis assay histone associated dna fragmentation
    Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and <t>apoptosis</t> was estimated using <t>ELISA</t> cell death assay by measuring histone associated <t>DNA</t> fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P
    Enzyme Linked Immunosorbent Assay Elisa Apoptosis Assay Histone Associated Dna Fragmentation, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche h2b mrfp h3 pa gfp expressing cells
    Labelling individual human interphase chromosomes in live cells. A) Activation of H3 <t>PA-GFP</t> (green) on a single chromosome in mitosis. Using <t>H2B</t> <t>mRFP,</t> mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm 3 ), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.
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    Roche hela human cervical carcinoma cells
    APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity ( A ) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, <t>HeLa,</t> HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. ( B ) HeLa cells were <t>transfected</t> for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. ( C ) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.
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    Roche h2b rfp
    Cell divisions within ndr2 -expressing explants. ( A ) Manually annotated cell divisions (per explant) within four live explants from WT embryos co-injected with <t>H2B-RFP</t> and 10 pg ndr2 RNA. Measurements were taken at 5 min intervals from 7.5 to 11 hpf. Boxes represent the 25 th to 75 th percentiles, whiskers are minimum and maximum vales, bars are median values. ( B ) Left: representative image of cell divisions (arrowheads) within an explant co-expressing H2B-RFP and ndr2 . The inset is enlarged from the region in the yellow square. Right: radial histogram of locations of all cell divisions (with respect to the center of each explant) detected within four ndr2- expressing explants between 7.5 and 11 hpf, with 90°/270° representing the axis of extension.
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    Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and apoptosis was estimated using ELISA cell death assay by measuring histone associated DNA fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P

    Journal: BMC Medicine

    Article Title: Antitumor activity of phenethyl isothiocyanate in HER2-positive breast cancer models

    doi: 10.1186/1741-7015-10-80

    Figure Lengend Snippet: Effect of phenethyl isothiocyanate (PEITC) on MDA-MB-231 cells expressing high HER2 (HH) . (A) Comparative effect of PEITC treatment on MDA-MB-231 cells with stable overexpression of HER2 relative to parent cells and vector control cells. Cells were treated for 24 h with 10 μM PEITC, and whole lysate was analyzed by western blotting for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Y705) and cleavage of poly-ADP ribose polymerase (PARP). (B) MDA-MB-231, vector control cells and high HER2 cells were treated with 10 μM of PEITC for 24 h and apoptosis was estimated using ELISA cell death assay by measuring histone associated DNA fragments. (C) MDA-MB-231 (HH) cells were treated with different concentrations of PEITC for 24 h, and the whole cell lysates were analyzed by western blotting. (D) MDA-MB-231 (HH) cells were treated with PEITC for 24, 48 and 72 h with increasing concentrations of PEITC and cell survival was measured by sulforhodamine B assay. The figures are representative of at least three independent experiments with eight replicates. * Statistically different compared with control ( P

    Article Snippet: Enzyme-linked immunosorbent assay (ELISA) apoptosis assay Histone associated DNA fragmentation during apoptosis was analyzed using an ELISA kit (Roche Applied Science, Indianapolis, IN, USA).

    Techniques: Multiple Displacement Amplification, Expressing, Over Expression, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Sulforhodamine B Assay

    Labelling individual human interphase chromosomes in live cells. A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm 3 ), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.

    Journal: PLoS ONE

    Article Title: Stable Morphology, but Dynamic Internal Reorganisation, of Interphase Human Chromosomes in Living Cells

    doi: 10.1371/journal.pone.0011560

    Figure Lengend Snippet: Labelling individual human interphase chromosomes in live cells. A) Activation of H3 PA-GFP (green) on a single chromosome in mitosis. Using H2B mRFP, mitotic cell with separated chromosomes were identified and separated chromosomes were scanned using a 405 nm laser. A z-stack was used to confirm single chromosome labelling (right image, projection of 3D stack, 5 µm bar). B–E) Sample maximal projections of different pairs of daughter nuclei (red) showing single interphase chromosomes (green). Bar 15 µm. Images captured between 1 and 4 hours after mitosis. F) Quantification of decondensation of mitotic chromosomes into 4 h interphase chromosomes. Box plots show data range of chromosome volumes (µm 3 ), the two mid-quartiles, and the median. Mitotic chromosomes n = 20, interphase n = 39.

    Article Snippet: For evaluating TSA treatment we probed Western blots of control and TSA-treated extracts of H2B-mRFP/H3-PA-GFP expressing cells with α-mouse anti-GFP (Roche # 11814460001), a α-rabbit H3 acetyl antibody, H3K9Ac and H3K14Ac (all acetylation antibodies from Upstate).

    Techniques: Activation Assay

    APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity ( A ) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, HeLa, HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. ( B ) HeLa cells were transfected for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. ( C ) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.

    Journal: Biochemical Journal

    Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    doi: 10.1042/BJ20090086

    Figure Lengend Snippet: APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity ( A ) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, HeLa, HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. ( B ) HeLa cells were transfected for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. ( C ) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.

    Article Snippet: Plasmid, siRNA and esiRNA transfection For microscopy analysis, HeLa human cervical carcinoma cells were transfected with 0.2 μg of plasmid DNA in 24-well plates using FuGENE™ reagent (Roche) and fixed 48 h after transfection.

    Techniques: Activity Assay, Western Blot, Transfection, Immunoprecipitation

    APPL1 overexpression affects the composition of HDAC1-containing NuRD complex and the expression of HDAC1 target p21 WAF1/CIP ( A ) APPL1 overexpression impairs the interactions of HDAC1 with other NuRD subunits. HEK-293 cells overexpressing untagged APPL1 at moderate or high levels (m.o., moderate overexpression of pAPPL1; h.o., high overexpression of pAPPL1) were subjected to immunoprecipitation (IP) with anti-HDAC1 or non-specific rabbit (IgG) antibodies. Immunoprecipitates along with 10% of the extracts used (input, right panel) were tested by immunoblotting for the presence of several NuRD subunits, as indicated. Some non-specific binding of APPL1 to IgG-covered Protein G beads is marked with an asterisk. ( B ) APPL1 overexpression reduces the association of HDAC1 with other NuRD components in the nuclear fraction. HDAC1 was immunoprecipitated from the nuclear extracts of HEK-293 cells with endogenous (vector) or overexpressed APPL1. Immunoprecipitates and 5% of the starting material (input, right panel) were blotted for the presence of the indicated NuRD components. ( C ) APPL1 influences the level of HDAC1 target gene product p21 WAF1/CIP1 . The level of p21 WAF1/CIP1 expression was analysed by Western blotting using anti-p21 antibody in extracts of cells with overexpression or silenced expression of APPL1. Left panel: Extracts of HEK-293 cells overexpressing APPL1 (either untagged, pAPPL1, or MYC-tagged, pAPPL1–MYC) or transfected with a control vector for 48 h were immunoblotted as indicated. No efficient overexpression of APPL1 could be achieved in HeLa cells. Right panel: APPL1 expression was reduced by esiRNA against APPL1 in HEK-293 or HeLa cells, using esiRNA against luciferase (luc) as a specificity control. Transfections with esiRNA were performed for 72 h (HEK-293) or 48 h (HeLa). The resulting extracts were immunoblotted against APPL1, Myc and p21. GAPDH was included as a loading control.

    Journal: Biochemical Journal

    Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    doi: 10.1042/BJ20090086

    Figure Lengend Snippet: APPL1 overexpression affects the composition of HDAC1-containing NuRD complex and the expression of HDAC1 target p21 WAF1/CIP ( A ) APPL1 overexpression impairs the interactions of HDAC1 with other NuRD subunits. HEK-293 cells overexpressing untagged APPL1 at moderate or high levels (m.o., moderate overexpression of pAPPL1; h.o., high overexpression of pAPPL1) were subjected to immunoprecipitation (IP) with anti-HDAC1 or non-specific rabbit (IgG) antibodies. Immunoprecipitates along with 10% of the extracts used (input, right panel) were tested by immunoblotting for the presence of several NuRD subunits, as indicated. Some non-specific binding of APPL1 to IgG-covered Protein G beads is marked with an asterisk. ( B ) APPL1 overexpression reduces the association of HDAC1 with other NuRD components in the nuclear fraction. HDAC1 was immunoprecipitated from the nuclear extracts of HEK-293 cells with endogenous (vector) or overexpressed APPL1. Immunoprecipitates and 5% of the starting material (input, right panel) were blotted for the presence of the indicated NuRD components. ( C ) APPL1 influences the level of HDAC1 target gene product p21 WAF1/CIP1 . The level of p21 WAF1/CIP1 expression was analysed by Western blotting using anti-p21 antibody in extracts of cells with overexpression or silenced expression of APPL1. Left panel: Extracts of HEK-293 cells overexpressing APPL1 (either untagged, pAPPL1, or MYC-tagged, pAPPL1–MYC) or transfected with a control vector for 48 h were immunoblotted as indicated. No efficient overexpression of APPL1 could be achieved in HeLa cells. Right panel: APPL1 expression was reduced by esiRNA against APPL1 in HEK-293 or HeLa cells, using esiRNA against luciferase (luc) as a specificity control. Transfections with esiRNA were performed for 72 h (HEK-293) or 48 h (HeLa). The resulting extracts were immunoblotted against APPL1, Myc and p21. GAPDH was included as a loading control.

    Article Snippet: Plasmid, siRNA and esiRNA transfection For microscopy analysis, HeLa human cervical carcinoma cells were transfected with 0.2 μg of plasmid DNA in 24-well plates using FuGENE™ reagent (Roche) and fixed 48 h after transfection.

    Techniques: Over Expression, Expressing, Immunoprecipitation, Binding Assay, Plasmid Preparation, Western Blot, Transfection, esiRNA, Luciferase

    HDAC2 is critical for binding of APPL1 to the NuRD complex ( A and B ) Extracts from HeLa cells transfected for 72 h with two (a, b) or three (a, b, c) different siRNA oligonucleotides per gene against: HDAC1, HDAC2, MTA2, RbAp48 and RbAp46 or non-specific siRNA (Φ) were subjected to immunoprecipitation (IP) using: ( A ) anti-APPL1 antibody; ( B ) anti-HDAC1 antibody (left panel) or anti-MTA2 antibody (right panel). Non-specific antibodies (IgG) were used as controls. Input indicates 10% of total cell extracts used for immunoprecipitation. Immunoprecipitates and input extracts were analysed by Western blotting using different antibodies as indicated. ( C ) To verify the direct interactions between APPL1 and HDAC1 or HDAC2, in vitro translated HDAC1–FLAG and untagged HDAC2 were subjected to GST pull-down assay using GST alone (Φ) or GST fused to the N- or C-terminal parts of APPL1 (APPL1-N or APPL1-C, respectively). Input indicates 10% of in vitro translated material used for the pull-down assay. Bound proteins were analysed by Western blotting using anti-HDAC1 and anti-HDAC2 antibodies. ND, not determined.

    Journal: Biochemical Journal

    Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    doi: 10.1042/BJ20090086

    Figure Lengend Snippet: HDAC2 is critical for binding of APPL1 to the NuRD complex ( A and B ) Extracts from HeLa cells transfected for 72 h with two (a, b) or three (a, b, c) different siRNA oligonucleotides per gene against: HDAC1, HDAC2, MTA2, RbAp48 and RbAp46 or non-specific siRNA (Φ) were subjected to immunoprecipitation (IP) using: ( A ) anti-APPL1 antibody; ( B ) anti-HDAC1 antibody (left panel) or anti-MTA2 antibody (right panel). Non-specific antibodies (IgG) were used as controls. Input indicates 10% of total cell extracts used for immunoprecipitation. Immunoprecipitates and input extracts were analysed by Western blotting using different antibodies as indicated. ( C ) To verify the direct interactions between APPL1 and HDAC1 or HDAC2, in vitro translated HDAC1–FLAG and untagged HDAC2 were subjected to GST pull-down assay using GST alone (Φ) or GST fused to the N- or C-terminal parts of APPL1 (APPL1-N or APPL1-C, respectively). Input indicates 10% of in vitro translated material used for the pull-down assay. Bound proteins were analysed by Western blotting using anti-HDAC1 and anti-HDAC2 antibodies. ND, not determined.

    Article Snippet: Plasmid, siRNA and esiRNA transfection For microscopy analysis, HeLa human cervical carcinoma cells were transfected with 0.2 μg of plasmid DNA in 24-well plates using FuGENE™ reagent (Roche) and fixed 48 h after transfection.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, In Vitro, Pull Down Assay

    The interactions with NuRD affect cellular distribution of APPL1 ( A ) APPL1 protein levels do not depend on HDAC1 or HDAC2. Extracts of HeLa cells with reduced HDAC1 or HDAC2 levels by siRNA [two different oligonucleotides (a, b) per gene or non-specific oligo (Φ), transfected for 72 h; top panel] and HEK-293 cells transfected for 48 h with plasmids encoding FLAG-tagged HDAC1 (pHDAC1–FLAG), HDAC2 (pHDAC2–FLAG) or with a control vector (bottom panel), were analysed by Western blotting using anti-APPL1 antibodies. To demonstrate the efficiency of silencing or overexpression, extracts were probed using anti-HDAC1 and anti-HDAC2 antibodies. GAPDH was included as a loading control. ( B ) Microscopy-based analysis of APPL1 nuclear localization upon silencing of HDAC1 or HDAC2. HeLa cells were transfected with siRNA oligonucleotides: one non-specific (Φ) and two specific per gene (a and b) against HDAC1 and HDAC2 for 72 h, followed by fixation and immunostaining for APPL1. Acquired microscopic images were analysed by Metamorph software and the average pixel intensities corresponding to APPL1 in the nuclei (as visualized by Hoechst staining, not shown) were calculated. The results of a representative experiment are shown in the graph. The values are normalized with respect to the average pixel intensity of nuclear APPL1 in cells transfected with non-specific siRNA, assigned one arbitrary unit. Error bars indicate standard error (minimum 100 cells from each transfection were used for the analysis). The results were statistically analysed by GraphPad Prism4 software, and the values obtained for each knockdown experiment were significantly different from the control at P

    Journal: Biochemical Journal

    Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    doi: 10.1042/BJ20090086

    Figure Lengend Snippet: The interactions with NuRD affect cellular distribution of APPL1 ( A ) APPL1 protein levels do not depend on HDAC1 or HDAC2. Extracts of HeLa cells with reduced HDAC1 or HDAC2 levels by siRNA [two different oligonucleotides (a, b) per gene or non-specific oligo (Φ), transfected for 72 h; top panel] and HEK-293 cells transfected for 48 h with plasmids encoding FLAG-tagged HDAC1 (pHDAC1–FLAG), HDAC2 (pHDAC2–FLAG) or with a control vector (bottom panel), were analysed by Western blotting using anti-APPL1 antibodies. To demonstrate the efficiency of silencing or overexpression, extracts were probed using anti-HDAC1 and anti-HDAC2 antibodies. GAPDH was included as a loading control. ( B ) Microscopy-based analysis of APPL1 nuclear localization upon silencing of HDAC1 or HDAC2. HeLa cells were transfected with siRNA oligonucleotides: one non-specific (Φ) and two specific per gene (a and b) against HDAC1 and HDAC2 for 72 h, followed by fixation and immunostaining for APPL1. Acquired microscopic images were analysed by Metamorph software and the average pixel intensities corresponding to APPL1 in the nuclei (as visualized by Hoechst staining, not shown) were calculated. The results of a representative experiment are shown in the graph. The values are normalized with respect to the average pixel intensity of nuclear APPL1 in cells transfected with non-specific siRNA, assigned one arbitrary unit. Error bars indicate standard error (minimum 100 cells from each transfection were used for the analysis). The results were statistically analysed by GraphPad Prism4 software, and the values obtained for each knockdown experiment were significantly different from the control at P

    Article Snippet: Plasmid, siRNA and esiRNA transfection For microscopy analysis, HeLa human cervical carcinoma cells were transfected with 0.2 μg of plasmid DNA in 24-well plates using FuGENE™ reagent (Roche) and fixed 48 h after transfection.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Over Expression, Microscopy, Immunostaining, Software, Staining

    Deacetylase activity detected in APPL1 complexes derives mainly from HDAC2 HDAC enzymatic activity was measured using a fluorimetric method (as described in the Experimental section) in immunoprecipitates from HeLa cells. ( A ) APPL1 binds active HDACs from class I or II. The APPL1 immunoprecipitate (IP) was divided into three equal parts: one left untreated and the other two treated with HDAC inhibitors: 1 mM nicotinamide and 1 μM TSA. Non-specific rabbit IgG was used as a control. ( B ) HeLa cells were silenced for HDAC1, HDAC2 and MTA2 [using two (a, b) different siRNA oligonucleotides per gene or non-specific siRNA Φ] prior to immunoprecipitation using APPL1 antibodies or non-specific rabbit IgG. The same extracts as presented in Figure 1 (A) were used (one third of the immunoprecipitates was measured in the HDAC activity assay, two thirds blotted as shown in Figure 1 A). The intensity of fluorescence emitted by the deacetylated substrate is expressed in arbitrary units in ( A ) and ( B ).

    Journal: Biochemical Journal

    Article Title: Functional characterization of the interactions between endosomal adaptor protein APPL1 and the NuRD co-repressor complex

    doi: 10.1042/BJ20090086

    Figure Lengend Snippet: Deacetylase activity detected in APPL1 complexes derives mainly from HDAC2 HDAC enzymatic activity was measured using a fluorimetric method (as described in the Experimental section) in immunoprecipitates from HeLa cells. ( A ) APPL1 binds active HDACs from class I or II. The APPL1 immunoprecipitate (IP) was divided into three equal parts: one left untreated and the other two treated with HDAC inhibitors: 1 mM nicotinamide and 1 μM TSA. Non-specific rabbit IgG was used as a control. ( B ) HeLa cells were silenced for HDAC1, HDAC2 and MTA2 [using two (a, b) different siRNA oligonucleotides per gene or non-specific siRNA Φ] prior to immunoprecipitation using APPL1 antibodies or non-specific rabbit IgG. The same extracts as presented in Figure 1 (A) were used (one third of the immunoprecipitates was measured in the HDAC activity assay, two thirds blotted as shown in Figure 1 A). The intensity of fluorescence emitted by the deacetylated substrate is expressed in arbitrary units in ( A ) and ( B ).

    Article Snippet: Plasmid, siRNA and esiRNA transfection For microscopy analysis, HeLa human cervical carcinoma cells were transfected with 0.2 μg of plasmid DNA in 24-well plates using FuGENE™ reagent (Roche) and fixed 48 h after transfection.

    Techniques: Histone Deacetylase Assay, Activity Assay, Immunoprecipitation, HDAC Activity Assay, Fluorescence

    Cell divisions within ndr2 -expressing explants. ( A ) Manually annotated cell divisions (per explant) within four live explants from WT embryos co-injected with H2B-RFP and 10 pg ndr2 RNA. Measurements were taken at 5 min intervals from 7.5 to 11 hpf. Boxes represent the 25 th to 75 th percentiles, whiskers are minimum and maximum vales, bars are median values. ( B ) Left: representative image of cell divisions (arrowheads) within an explant co-expressing H2B-RFP and ndr2 . The inset is enlarged from the region in the yellow square. Right: radial histogram of locations of all cell divisions (with respect to the center of each explant) detected within four ndr2- expressing explants between 7.5 and 11 hpf, with 90°/270° representing the axis of extension.

    Journal: eLife

    Article Title: Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence and extension gastrulation movements

    doi: 10.7554/eLife.54445

    Figure Lengend Snippet: Cell divisions within ndr2 -expressing explants. ( A ) Manually annotated cell divisions (per explant) within four live explants from WT embryos co-injected with H2B-RFP and 10 pg ndr2 RNA. Measurements were taken at 5 min intervals from 7.5 to 11 hpf. Boxes represent the 25 th to 75 th percentiles, whiskers are minimum and maximum vales, bars are median values. ( B ) Left: representative image of cell divisions (arrowheads) within an explant co-expressing H2B-RFP and ndr2 . The inset is enlarged from the region in the yellow square. Right: radial histogram of locations of all cell divisions (with respect to the center of each explant) detected within four ndr2- expressing explants between 7.5 and 11 hpf, with 90°/270° representing the axis of extension.

    Article Snippet: Blastoderm explants Embryos were injected with ndr2 , H2B-RFP, and/or membrane GFP RNA (and MOs) at the one-cell stage as described above, or left uninjected, then dechorionated using Pronase (Roche).

    Techniques: Expressing, Injection