histone h1 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore histone h4
    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX
    Histone H4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h4/product/Millipore
    Average 99 stars, based on 789 article reviews
    Price from $9.99 to $1999.99
    histone h4 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    96
    Millipore soluble histone h1
    The cdc2/puc1 kinase can efficiently phosphorylate rum1 in vitro and is not inhibited by rum1. (A) Wild-type cells were grown to midexponential phase in minimal medium. Two milligrams of total protein extracts were immunoprecipitated with anti-cdc2, anti-cig1, and anti-puc1 antibodies. Protein kinase activity was measured with the use of rum1, rum1-A58A62 (A58A62), and <t>histone</t> H1 (H1) as substrates. The phosphorylated products were separated by 14% SDS-PAGE and exposed to autoradiography. Cdc2 immunocomplexes could phosphorylate p25 rum1 as efficiently as they could phosphorylate histone H1. Rum1 phosphorylation induced a band shift from 34 to 36 kDa that was not observed in the mutant rum1-A58A62. Cig1 and puc1 immunocomplexes induced a similar band shift to cdc2. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2, anti-cig1, and anti-puc1 antibodies. The immunoprecipitates were preincubated with different concentrations of rum1 protein and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ , cig2Δ , and puc1Δ were immunoprecipitated with anti-cig1, anti-cig2, and anti-puc1 antibodies and assayed for histone H1 kinase activity. IP, immunoprecipitate.
    Soluble Histone H1, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/soluble histone h1/product/Millipore
    Average 96 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    soluble histone h1 - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    99
    Millipore histone h3 bioworld
    The cdc2/puc1 kinase can efficiently phosphorylate rum1 in vitro and is not inhibited by rum1. (A) Wild-type cells were grown to midexponential phase in minimal medium. Two milligrams of total protein extracts were immunoprecipitated with anti-cdc2, anti-cig1, and anti-puc1 antibodies. Protein kinase activity was measured with the use of rum1, rum1-A58A62 (A58A62), and <t>histone</t> H1 (H1) as substrates. The phosphorylated products were separated by 14% SDS-PAGE and exposed to autoradiography. Cdc2 immunocomplexes could phosphorylate p25 rum1 as efficiently as they could phosphorylate histone H1. Rum1 phosphorylation induced a band shift from 34 to 36 kDa that was not observed in the mutant rum1-A58A62. Cig1 and puc1 immunocomplexes induced a similar band shift to cdc2. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2, anti-cig1, and anti-puc1 antibodies. The immunoprecipitates were preincubated with different concentrations of rum1 protein and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ , cig2Δ , and puc1Δ were immunoprecipitated with anti-cig1, anti-cig2, and anti-puc1 antibodies and assayed for histone H1 kinase activity. IP, immunoprecipitate.
    Histone H3 Bioworld, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3 bioworld/product/Millipore
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    histone h3 bioworld - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    92
    Roche histone h1
    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 <t>histone</t> 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.
    Histone H1, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 835 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Roche
    Average 92 stars, based on 835 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Boehringer Mannheim histone h1
    FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated <t>histone</t> H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.
    Histone H1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Boehringer Mannheim
    Average 92 stars, based on 436 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology histone h3
    Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated <t>histone</t> H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
    Histone H3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1550 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1550 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    90
    Merck & Co histone h1
    GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for <t>histone</t> H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), histone H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.
    Histone H1, supplied by Merck & Co, used in various techniques. Bioz Stars score: 90/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Merck & Co
    Average 90 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    92
    Upstate Biotechnology Inc histone h4
    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular <t>histone</t> H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.
    Histone H4, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h4/product/Upstate Biotechnology Inc
    Average 92 stars, based on 621 article reviews
    Price from $9.99 to $1999.99
    histone h4 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    Promega histone h1
    The dCK–Cdk1 interaction inhibits Cdk1 activity both in vitro and in vivo . ( A ) Cdk1 kinase activity was measured by an in vitro kinase assay using the recombinant Cdk1/CyclinA2 complex incubated with 1 μg of <t>Histone</t> H1 as substrate in the presence of 50 µM ATP. Recombinant dCKs at indicated doses were added into the kinase reaction, and the luminescence signal indicative of Cdk1 activity was recorded. The average and standard deviations of at least triplicate samples are shown. ( B ) Isogenic HeLa cell lines with control or dCK shRNA were treated with mock or IR (6 Gy). Two hours after IR, total cell lysates were obtained and subjected to western blot analysis using indicated antibodies. ( C ) HeLa cells transiently transfected with vector, flag-tagged wild-type, S74A or S74E were irradiated (6 Gy). Total cell lysates were obtained and subjected to western blot with indicated antibodies.
    Histone H1, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Promega
    Average 93 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    Bioworld Antibodies histone h1
    Western blot analysis of GR, p-CREB and Sp1 in liver nuclear protein extracts of LW and EHL pigets. <t>Histone</t> H1 was used as a loading control. The nuclear contents of the transcription factors are presented as the fold change relative to LW. Blank bars represent Large White (LW) piglets; filled bars represent Erhualian (EHL) piglets. Values are presented as mean ± SEM. ** donates P
    Histone H1, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Bioworld Antibodies
    Average 92 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    Millipore anti histone h3
    β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and <t>histone</t> H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.
    Anti Histone H3, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti histone h3/product/Millipore
    Average 93 stars, based on 845 article reviews
    Price from $9.99 to $1999.99
    anti histone h3 - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    Abcam histone h3
    Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of <t>histone</t> H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .
    Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3/product/Abcam
    Average 94 stars, based on 6614 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    93
    SignalChem histone h1
    Amino acid sequence and phosphorylated sites of bovine histone H1.2 as determined by mass spectrometry. Lysine residues are marked red, and those identified as targets for the phosphorylation by phosphoramidate (i.e. seven residues) are highlighted. Out of the 305 peptides that were used to identify the protein, 14 peptides were reported to contain phospholysine.
    Histone H1, supplied by SignalChem, used in various techniques. Bioz Stars score: 93/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/SignalChem
    Average 93 stars, based on 235 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    Echelon Biosciences histone h1
    A typical response of ENaC to exogenous PI(3,4,5)P 3 . Shown is a representative current trace from an excised, outside-out patch (V p = 0 mV) formed from a CHO cell expressing wild-type ENaC before and after addition of 20 μM exogenous diC8 PI(3,4,5)P 3 . PI(3,4,5)P 3 added to the bathing solution in the presence of <t>histone</t> H1 carrier. Amiloride subsequently added to the bath solution toward the end of the experiment. This representative patch contains, at least, five ENaC. Shown at top is a continuous trace. Shown below (left) at an expanded timescale are regions of the trace before (1. control; middle) and after (2. PIP 3 ; bottom) addition of exogenous phosphoinositide. Respective all-point histograms for the regions shown at an expanded time-scale are to the right. All other conditions the same as Fig. 3 A .
    Histone H1, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Echelon Biosciences
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Proteintech histone h1
    Effect of the expression of UL23 on the distribution of Nmi and STAT1 in nuclear and cytoplasmic fractions. (A) U251 (lanes 2 and 4) and U251-UL23 cells (lanes 1 and 3) were treated with IFN-γ (1000 U/ml) and harvested at 36 hours post-treatment. The IFN-γ treated U251 cells were infected with HCMV Towne BAC  (lanes 5 and 7) or ΔUL23 (lanes 6 and 8) (MOI = 1) at 12 hours post-treatment and harvested at 24 hours postinfection. The harvested cells were separated into nuclear and cytoplasmic fractions. Equivalent amounts of each fraction were analyzed by immunoblotting with anti-UL23, anti-Nmi, and anti-STAT1. The purity of the nuclear and cytoplasmic fractions was assayed by immunoblotting with anti-histone H1 and anti-actin, respectively. The membranes were reacted with antibodies and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [  60 ,  64 ]. The protein levels of Nmi (B) and STAT1 (C) in the nuclei and cytoplasm of different cells that were mock-infected or infected with different viruses were quantified. The experiments were repeated three times. The standard deviation is indicated by the error bar.
    Histone H1, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Proteintech
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Boehringer Ingelheim histone h1
    Chromosome segregation in meiosis I is abortive in Gwl-inhibited oocytes. (A and B) Immature starfish oocytes were first injected with HiLyte 488–labeled tubulin (green) and Alexa 568–labeled <t>histone</t> H1 (magenta), and then injected with neutralizing anti-Gwl antibody (anti-Gwl) or control IgG (cont. IgG) along with ZZ-IBB (that can deliver cytoplasmically injected IgG into the nucleus) or uninjected (None). After 1-MeAde addition, live-cell images were obtained using a confocal microscope to monitor formation of the meiosis I spindle. Gwl-inhibited oocytes failed to properly segregate homologous chromosomes (A). This phenotype may be classified into three types, although they overlapped in some cases: lagging (#1, magenta), congressed (#2, yellow), or scattered (#3, blue) chromosomes along with frequently multipolar spindles (asterisks). However, further coinjection into immature oocytes with Arpp19 that had been in vitro thiophosphorylated by Gwl (pS106-Arpp19), but not with wild-type Arpp19 (wt-Arpp19), restored homologous chromosome segregation (B). Time after 1-MeAde addition is indicated in each frame. Arrow, chromosomes; arrowhead, the first polar body. Bars, 5 µm. (C) Quantification of chromosome segregation failure displayed in A and B. Each color corresponds to #1, #2, and #3 in A and B, respectively. Numbers of independent experiments (more than three females) are indicated at each bar.
    Histone H1, supplied by Boehringer Ingelheim, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Boehringer Ingelheim
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Fisher Scientific histone h1
    Chromosome segregation in meiosis I is abortive in Gwl-inhibited oocytes. (A and B) Immature starfish oocytes were first injected with HiLyte 488–labeled tubulin (green) and Alexa 568–labeled <t>histone</t> H1 (magenta), and then injected with neutralizing anti-Gwl antibody (anti-Gwl) or control IgG (cont. IgG) along with ZZ-IBB (that can deliver cytoplasmically injected IgG into the nucleus) or uninjected (None). After 1-MeAde addition, live-cell images were obtained using a confocal microscope to monitor formation of the meiosis I spindle. Gwl-inhibited oocytes failed to properly segregate homologous chromosomes (A). This phenotype may be classified into three types, although they overlapped in some cases: lagging (#1, magenta), congressed (#2, yellow), or scattered (#3, blue) chromosomes along with frequently multipolar spindles (asterisks). However, further coinjection into immature oocytes with Arpp19 that had been in vitro thiophosphorylated by Gwl (pS106-Arpp19), but not with wild-type Arpp19 (wt-Arpp19), restored homologous chromosome segregation (B). Time after 1-MeAde addition is indicated in each frame. Arrow, chromosomes; arrowhead, the first polar body. Bars, 5 µm. (C) Quantification of chromosome segregation failure displayed in A and B. Each color corresponds to #1, #2, and #3 in A and B, respectively. Numbers of independent experiments (more than three females) are indicated at each bar.
    Histone H1, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/Fisher Scientific
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    GeneTex histone h3
    Nuclear translocation of Nrf2 leads to drug resistance under hypoxia A-C. MCF7 cells were exposed to hypoxia for 0, 4, 8, and 24 hours. (A) Nrf2 localization was determined by immunocytochemistry (ICC) with an anti-Nrf2 antibody (green fluorescence). Nuclear location was determined by DAPI staining (blue fluorescence). (B, C) Total proteins and cytosolic (C)/nuclear (N) proteins were extracted. The protein levels of Hif-1α, Nrf2, GAPDH, and <t>Histone</t> H3 were detected by western blotting. GAPDH was the loading control for the cytosolic fraction, and Histone H3 was the loading control for the nuclear fraction. D. MCF7 cells were transfected with pARE-CMV and pRL-CMV plasmids. After transfection, the cells were exposed to hypoxia for 0, 4, 8, and 24 hours, and the firefly and Renilla luminescence activities were detected by Dual-Glo luciferase assay. The luciferase activity was represented by firefly luminescence normalized with Renilla luminescence. N=3, *, P
    Histone H3, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3/product/GeneTex
    Average 92 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    Bio-Rad histone h3
    ATPase-dependent activity of the Mms21 SUMO ligase. A . Growth test of GALp-SMC5 cells expressing wild-type SMC5 , smc5(K75I) , or smc5(D1014A) from a centromeric vector in plates containing galactose ( GALp ON) or glucose ( GALp OFF). B . Mms21-3HA was immunoprecipitated from exponentially growing cells transformed with the indicated SMC5 -expressing centromeric plasmids to test the Smc5-Mms21 interaction; wt = wild type; KI = smc5(K75I) ; DA = smc5(D1014A) . C . Sumoylation analysis of ATPase-defective Smc5-9myc proteins. HF-SUMO pull-down analysis in wild-type cells transformed with plasmids expressing the indicated SMC5 alleles. D . Sumoylation analysis of Nse4-6HA in smc5 ATPase mutant cells. HF-SUMO pull-down analysis in GALp-SMC5 NSE4-6HA cells expressing the indicated SMC5 alleles. Cells were shifted to glucose 6 h before collection to repress the endogenous SMC5 gene. E . Sumoylation analysis of cohesin in smc5 ATPase mutant cells. HF-SUMO pull down from cells of the indicated genotype (wt, mms21ΔC and GALp-SMC5 ), carrying a C-terminal 6HA tag on SMC1 , and expressing or not an ectopic copy of SMC5-9myc (WT) or smc5(K75I)-9myc (KI) allele; where indicated, cells were treated with MMS 0,02% for 1 h (MMS) before collection. F . Chromatin fractionation assay from GALp-SMC5 MMS21-6HA cells expressing an ectopic 9myc-tagged copy of the indicated SMC5 alleles, collected 6 h after shift to glucose to deplete the endogenous Smc5 protein. Controls for a chromatin-bound protein <t>(histone</t> H3), nuclear soluble (Rpd3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown; WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction. In C–E, arrow points to unmodified Smc5, Nse4, or Smc1 proteins, and vertical bars to their sumoylated forms.
    Histone H3, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h3/product/Bio-Rad
    Average 93 stars, based on 130 article reviews
    Price from $9.99 to $1999.99
    histone h3 - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    92
    ALPCO histone h1
    ATPase-dependent activity of the Mms21 SUMO ligase. A . Growth test of GALp-SMC5 cells expressing wild-type SMC5 , smc5(K75I) , or smc5(D1014A) from a centromeric vector in plates containing galactose ( GALp ON) or glucose ( GALp OFF). B . Mms21-3HA was immunoprecipitated from exponentially growing cells transformed with the indicated SMC5 -expressing centromeric plasmids to test the Smc5-Mms21 interaction; wt = wild type; KI = smc5(K75I) ; DA = smc5(D1014A) . C . Sumoylation analysis of ATPase-defective Smc5-9myc proteins. HF-SUMO pull-down analysis in wild-type cells transformed with plasmids expressing the indicated SMC5 alleles. D . Sumoylation analysis of Nse4-6HA in smc5 ATPase mutant cells. HF-SUMO pull-down analysis in GALp-SMC5 NSE4-6HA cells expressing the indicated SMC5 alleles. Cells were shifted to glucose 6 h before collection to repress the endogenous SMC5 gene. E . Sumoylation analysis of cohesin in smc5 ATPase mutant cells. HF-SUMO pull down from cells of the indicated genotype (wt, mms21ΔC and GALp-SMC5 ), carrying a C-terminal 6HA tag on SMC1 , and expressing or not an ectopic copy of SMC5-9myc (WT) or smc5(K75I)-9myc (KI) allele; where indicated, cells were treated with MMS 0,02% for 1 h (MMS) before collection. F . Chromatin fractionation assay from GALp-SMC5 MMS21-6HA cells expressing an ectopic 9myc-tagged copy of the indicated SMC5 alleles, collected 6 h after shift to glucose to deplete the endogenous Smc5 protein. Controls for a chromatin-bound protein <t>(histone</t> H3), nuclear soluble (Rpd3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown; WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction. In C–E, arrow points to unmodified Smc5, Nse4, or Smc1 proteins, and vertical bars to their sumoylated forms.
    Histone H1, supplied by ALPCO, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h1/product/ALPCO
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    histone h1 - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: Apoptotic cells exhibit reduced staining for acetylated histone H4. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Staining, Labeling, Variant Assay

    HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Silencing of Fem1cR3 Gene Expression in the DBA/2J Mouse Precedes Retinal Ganglion Cell Death and Is Associated with Histone Deacetylase Activity

    doi: 10.1167/iovs.11-8872

    Figure Lengend Snippet: HDAC3 concentrates in nuclei of apoptotic cells. Immunofluorescent double labeling of cells in the ganglion cell layer of a DBA/2J mouse. Frozen sections were double labeled with antibodies against the histone variant γH2AX to identify dying cells,

    Article Snippet: Similarly, colocalization of acetylated histone H4 (AcH4) and γH2AX showed that normal-appearing (stage I) cells had robust AcH4 labeling ( ).

    Techniques: Labeling, Variant Assay

    The cdc2/puc1 kinase can efficiently phosphorylate rum1 in vitro and is not inhibited by rum1. (A) Wild-type cells were grown to midexponential phase in minimal medium. Two milligrams of total protein extracts were immunoprecipitated with anti-cdc2, anti-cig1, and anti-puc1 antibodies. Protein kinase activity was measured with the use of rum1, rum1-A58A62 (A58A62), and histone H1 (H1) as substrates. The phosphorylated products were separated by 14% SDS-PAGE and exposed to autoradiography. Cdc2 immunocomplexes could phosphorylate p25 rum1 as efficiently as they could phosphorylate histone H1. Rum1 phosphorylation induced a band shift from 34 to 36 kDa that was not observed in the mutant rum1-A58A62. Cig1 and puc1 immunocomplexes induced a similar band shift to cdc2. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2, anti-cig1, and anti-puc1 antibodies. The immunoprecipitates were preincubated with different concentrations of rum1 protein and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ , cig2Δ , and puc1Δ were immunoprecipitated with anti-cig1, anti-cig2, and anti-puc1 antibodies and assayed for histone H1 kinase activity. IP, immunoprecipitate.

    Journal: Molecular Biology of the Cell

    Article Title: The puc1 Cyclin Regulates the G1 Phase of the Fission Yeast Cell Cycle in Response to Cell Size

    doi:

    Figure Lengend Snippet: The cdc2/puc1 kinase can efficiently phosphorylate rum1 in vitro and is not inhibited by rum1. (A) Wild-type cells were grown to midexponential phase in minimal medium. Two milligrams of total protein extracts were immunoprecipitated with anti-cdc2, anti-cig1, and anti-puc1 antibodies. Protein kinase activity was measured with the use of rum1, rum1-A58A62 (A58A62), and histone H1 (H1) as substrates. The phosphorylated products were separated by 14% SDS-PAGE and exposed to autoradiography. Cdc2 immunocomplexes could phosphorylate p25 rum1 as efficiently as they could phosphorylate histone H1. Rum1 phosphorylation induced a band shift from 34 to 36 kDa that was not observed in the mutant rum1-A58A62. Cig1 and puc1 immunocomplexes induced a similar band shift to cdc2. (B) Wild-type fission yeast extracts were immunoprecipitated with anti-cdc13, anti-cig2, anti-cig1, and anti-puc1 antibodies. The immunoprecipitates were preincubated with different concentrations of rum1 protein and then assayed for histone H1 kinase activity. As negative controls (Δ), extracts of cig1Δ , cig2Δ , and puc1Δ were immunoprecipitated with anti-cig1, anti-cig2, and anti-puc1 antibodies and assayed for histone H1 kinase activity. IP, immunoprecipitate.

    Article Snippet: Immunoprecipitates (∼20 μl) were preincubated with different concentrations of purified rum1 protein for 5 min, diluted with 20 μl of homogenizing buffer containing 50 μM ATP, 0.5 mg/ml substrate histone H1 (Calbiochem, La Jolla, CA), and 40 μCi/ml [γ-32 P]ATP, and incubated at 25°C for 30 min.

    Techniques: In Vitro, Immunoprecipitation, Activity Assay, SDS Page, Autoradiography, Electrophoretic Mobility Shift Assay, Mutagenesis

    Rum1 protein persists for longer in the cig1Δ cig2Δ puc1Δ mutant than in the wild type. Wild-type and cig1Δ cig2Δ puc1Δ mutant cells were nitrogen starved for 8 h, and then nitrogen was added back to the culture. Samples were taken for protein extracts and flow cytometry at the indicated times. (A) Cdc13, rum1, and cdc2 protein levels in wild-type and cig1Δ cig2Δ puc1Δ cells. +N corresponds to cells growing in minimal medium. (B) Cdc2/cdc13 kinase assays. Cdc2/cdc13 complexes were immunoprecipitated with anti-cdc13 antibodies and assayed with the use of histone H1 as substrate. (C) Flow cytometry analysis of wild-type and cig1Δ cig2Δ puc1Δ mutant cells during the release from nitrogen starvation.

    Journal: Molecular Biology of the Cell

    Article Title: The puc1 Cyclin Regulates the G1 Phase of the Fission Yeast Cell Cycle in Response to Cell Size

    doi:

    Figure Lengend Snippet: Rum1 protein persists for longer in the cig1Δ cig2Δ puc1Δ mutant than in the wild type. Wild-type and cig1Δ cig2Δ puc1Δ mutant cells were nitrogen starved for 8 h, and then nitrogen was added back to the culture. Samples were taken for protein extracts and flow cytometry at the indicated times. (A) Cdc13, rum1, and cdc2 protein levels in wild-type and cig1Δ cig2Δ puc1Δ cells. +N corresponds to cells growing in minimal medium. (B) Cdc2/cdc13 kinase assays. Cdc2/cdc13 complexes were immunoprecipitated with anti-cdc13 antibodies and assayed with the use of histone H1 as substrate. (C) Flow cytometry analysis of wild-type and cig1Δ cig2Δ puc1Δ mutant cells during the release from nitrogen starvation.

    Article Snippet: Immunoprecipitates (∼20 μl) were preincubated with different concentrations of purified rum1 protein for 5 min, diluted with 20 μl of homogenizing buffer containing 50 μM ATP, 0.5 mg/ml substrate histone H1 (Calbiochem, La Jolla, CA), and 40 μCi/ml [γ-32 P]ATP, and incubated at 25°C for 30 min.

    Techniques: Mutagenesis, Flow Cytometry, Cytometry, Immunoprecipitation

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm032

    Figure Lengend 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.

    Article Snippet: Preparation of the protein samples The final composition of the storage buffers: KF− : 10 mM Tris pH 8, 0.5 mM EDTA, 100 µg/ml bovine serum albumin (BSA), 50% glycerol and 2 mM MgSO4 ; histone H1: 10 mM Tris pH 7.9, 20 mM NaCl, 0.1 mM PMSF for histone H1; NF-κB protein (p50 dimer): 25 mM Tris/HCl pH 8.0, 50 mM NaCl.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm032

    Figure Lengend 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.

    Article Snippet: Preparation of the protein samples The final composition of the storage buffers: KF− : 10 mM Tris pH 8, 0.5 mM EDTA, 100 µg/ml bovine serum albumin (BSA), 50% glycerol and 2 mM MgSO4 ; histone H1: 10 mM Tris pH 7.9, 20 mM NaCl, 0.1 mM PMSF for histone H1; NF-κB protein (p50 dimer): 25 mM Tris/HCl pH 8.0, 50 mM NaCl.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkm032

    Figure Lengend 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.

    Article Snippet: Preparation of the protein samples The final composition of the storage buffers: KF− : 10 mM Tris pH 8, 0.5 mM EDTA, 100 µg/ml bovine serum albumin (BSA), 50% glycerol and 2 mM MgSO4 ; histone H1: 10 mM Tris pH 7.9, 20 mM NaCl, 0.1 mM PMSF for histone H1; NF-κB protein (p50 dimer): 25 mM Tris/HCl pH 8.0, 50 mM NaCl.

    Techniques: Sequencing, Modification, Nucleic Acid Electrophoresis, Incubation

    FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

    Journal: Molecules (Basel, Switzerland)

    Article Title: Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin

    doi: 10.3390/molecules22030462

    Figure Lengend Snippet: FKA increases MPM-2 phosphorylation and Cdc kinase activity via inhibition of Cdc2 and Cdc25 phosphorylation and downregulation of Myt1 and Wee1 expression. ( A ) Cdc2 associated histone H1 kinase activity was decreased dose-dependently by FKA treatment for 24 h in SKBR3 cells; ( B – D ) SKBR3 cells were treated with indicated concentrations of FKA for 24 h. Phosphorylation levels of Cdc2, MPM-2, and Cdc25C as well as protein levels of cyclin B1, Myt1, and Wee1 were detected by Western blotting analysis. β-actin was detected as a loading control. A representative blot was shown from three independent experiments.

    Article Snippet: Phosphorylation of histone H1 was measured by incubating the beads with 40 μL of “hot” kinase solution [0.25 μL (2.5 μg) of histone H1, 0.5 μL of [γ-32 P] ATP, 0.5 μL of 0.1 mM ATP, and 38.75 μL of kinase buffer] for 30 min at 37 °C.

    Techniques: Activity Assay, Inhibition, Expressing, Western Blot

    Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

    Journal: International Journal of Environmental Research and Public Health

    Article Title: NNK, a Tobacco-Specific Carcinogen, Inhibits the Expression of Lysyl Oxidase, a Tumor Suppressor

    doi: 10.3390/ijerph120100064

    Figure Lengend Snippet: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 10 6 , sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

    Article Snippet: As shown in , in comparison to the internal control, the GAPDH fragment bound with the RNA-PolyII, the histone H3 acetylated at the tested region of the LOX promoter in NNK-treated cells was reduced to 74, 41, 11 and 5% of the control for cells treated with 10, 30, 100 and 300 µM NNK, respectively.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Isolation, Sonication, Immunoprecipitation, Amplification, Software

    GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), histone H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.

    Journal: Theranostics

    Article Title: Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis

    doi: 10.7150/thno.24173

    Figure Lengend Snippet: GO/CDDP triggered direct association of histones H1/H4 and CDDP with LC3. (A) SDS-PAGE analysis of LC3-immunocomplex from the lysate (Lys) and nuclear (Nuc) fractions of cells with or without GO/CDDP treatment. (B) Western blot analysis of proteins (~11 kDa and ~35 kDa) derived from the nuclear LC3-immunocomplex. (C) Immunofluorescence microscopy of cells with or without GO/CDDP treatment. The cells were immunolabeled with primary antibody for histone H1 or H4 and counter stained with DAPI. (D) Percentages of cells positive with total histone H1, phosphorylated histone H1 (p-histone H1), histone H4 and acetylated histone H4 (H4K16ac). (E) Amount of CDDP associated with nuclear LC3 immunocomplex. The nuclear LC3 immunocomplex was analyzed by ICP-MS to quantify the amount of platinum (Pt) in CDDP. The data represent mean ± s.d of at least 3 independent culture experiments.

    Article Snippet: LC/MS/MS analysis of these LC3-associated proteins ( Table -2 ) unraveled that the proteins were presumably histone H1 (~35 kDa) and histone H4 (~11 kDa).

    Techniques: SDS Page, Western Blot, Derivative Assay, Immunofluorescence, Microscopy, Immunolabeling, Staining, Mass Spectrometry

    Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Recovery of histone acetylation by overexpression of wt p300 HAT. HS27/vIRF cells were transfected with wt p300 (lane 3) or p300 ΔHAT mutant (lane 4). HS27/cDNA3 (lane 1) and HS27/vIRF (lane 2) were included as controls. Identical amounts of proteins from cell lysates were used for immunoblotting analysis with an antibody specific for the acetylated histone H4. The bottom panel shows the amount of cellular histone H4 protein in each lane, detected by an anti-H4 antibody. Arrows indicate the acetylated form of histone H4 (Ac-H4) or total histone H4 (H4). Numbers at left are molecular masses in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Over Expression, HAT Assay, Transfection, Mutagenesis

    Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Immunofluorescence test of in vivo histone H3 and H4 acetylation. HS27/cDNA3 and HS27/vIRF cells were stained with antibodies which specifically reacted with the acetylated forms of histones H3 and H4. Cells were visualized with Nomarski optics. Immunofluorescence testing was performed with a Leica confocal immunofluorescence microscope.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Immunofluorescence, In Vivo, Staining, Microscopy

    Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Inhibition of p300 HAT activity by vIRF. Recombinant baculovirus containing the flag-tagged p300, vIRF, or v-cyclin was used to purify each protein from insect cells. Purified p300 protein (30 nM) was mixed with [ 3 H]acetyl-CoA and histone H4 serving as substrates in the presence of increasing nanomolar amounts of vIRF or v-cyclin as indicated at the bottom of panel A. After 5 min, p300 HAT activity was measured by immunoblotting with an antibody specific for acetylated histone H4 (A) and quantitating radioactivity of 3 H-labeled histone H4 (B). In lane 7, p300 protein was first mixed with the substrates for 5 min, followed by incubation with vIRF protein (150 nM) for an additional 25 min. The bottom panel of panel A shows the amount of histone H4 protein used in each reaction, detected by an anti-H4 antibody. The values in panel B represent the averages of three independent experiments.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: Inhibition, HAT Assay, Activity Assay, Recombinant, Purification, Radioactivity, Labeling, Incubation

    Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of p300 Histone Acetyltransferase by Viral Interferon Regulatory Factor

    doi:

    Figure Lengend Snippet: Alteration of in vivo histone H3 and H4 acetylation by vIRF expression or butyric acid treatment. Identical amounts of proteins from HS27/cDNA3 cells (lanes 1 and 3) and HS27/vIRF cells (lanes 2 and 4) treated with butyric acid overnight (lanes 3 and 4) or mock treated (lanes 1 and 2) were used for immunoblotting analysis with antibodies specific for the acetylated histone H3 (A) or H4 (B). Arrows indicate acetylated histones H3 (Ac-H3) and H4 (Ac-H4). Numbers at left of each panel show sizes in kilodaltons.

    Article Snippet: Purified full-length p300 was mixed with histone H4 and 3 H-labeled acetyl-CoA in the presence or absence of purified vIRF protein.

    Techniques: In Vivo, Expressing

    The dCK–Cdk1 interaction inhibits Cdk1 activity both in vitro and in vivo . ( A ) Cdk1 kinase activity was measured by an in vitro kinase assay using the recombinant Cdk1/CyclinA2 complex incubated with 1 μg of Histone H1 as substrate in the presence of 50 µM ATP. Recombinant dCKs at indicated doses were added into the kinase reaction, and the luminescence signal indicative of Cdk1 activity was recorded. The average and standard deviations of at least triplicate samples are shown. ( B ) Isogenic HeLa cell lines with control or dCK shRNA were treated with mock or IR (6 Gy). Two hours after IR, total cell lysates were obtained and subjected to western blot analysis using indicated antibodies. ( C ) HeLa cells transiently transfected with vector, flag-tagged wild-type, S74A or S74E were irradiated (6 Gy). Total cell lysates were obtained and subjected to western blot with indicated antibodies.

    Journal: Nucleic Acids Research

    Article Title: Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

    doi: 10.1093/nar/gks707

    Figure Lengend Snippet: The dCK–Cdk1 interaction inhibits Cdk1 activity both in vitro and in vivo . ( A ) Cdk1 kinase activity was measured by an in vitro kinase assay using the recombinant Cdk1/CyclinA2 complex incubated with 1 μg of Histone H1 as substrate in the presence of 50 µM ATP. Recombinant dCKs at indicated doses were added into the kinase reaction, and the luminescence signal indicative of Cdk1 activity was recorded. The average and standard deviations of at least triplicate samples are shown. ( B ) Isogenic HeLa cell lines with control or dCK shRNA were treated with mock or IR (6 Gy). Two hours after IR, total cell lysates were obtained and subjected to western blot analysis using indicated antibodies. ( C ) HeLa cells transiently transfected with vector, flag-tagged wild-type, S74A or S74E were irradiated (6 Gy). Total cell lysates were obtained and subjected to western blot with indicated antibodies.

    Article Snippet: In vitro kinase assay For the Cdk1 in vitro kinase assay, recombinant dCK (Novus Biologicals, Littleton, CO, USA) and Cdk1/CyclinA2 (Promega, Madison, WI, USA) were incubated with 1 μg of Histone H1 (Promega) in the presence of 50 µM ATP in kinase buffer for 30 min at room temperature.

    Techniques: Activity Assay, In Vitro, In Vivo, Kinase Assay, Recombinant, Incubation, shRNA, Western Blot, Transfection, Plasmid Preparation, Irradiation

    ATM-mediated dCK Serine 74 phosphorylation is required for the IR-induced G2/M checkpoint. ( A ) dCK knock-down HeLa cells were re-introduced with vector control, dCK wild-type, dCK Serine 74 to alanine (S74A) mutation or Serine 74 to Glutamic Acid (S74E) mutation. The complementation effects using RT–PCR and western blot are shown. ( B ) The dCK complemented cell lines were treated with mock or IR (6 Gy). Two hours after treatment, cells were harvested and stained for Histone H3 Ser10 phosphorylation followed by flow cytometry. ( C ) Expression of flag-tagged dCK as compared to endogenous dCK in control shRNA and ATM shRNA cells. ( D ) HeLa cells with control or ATM shRNA were transiently transfected with vector, wild-type (WT), S74A or S74E of dCK and treated with IR (6 Gy). Two hours after treatment, cells were harvested and stained for Histone H3 Ser10 phosphorylation followed by flow cytometry analysis. The average and standard deviations of at least triplicate samples are shown. Statistic analysis was done by Student’s t -test; * P ≤ 0.05.

    Journal: Nucleic Acids Research

    Article Title: Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

    doi: 10.1093/nar/gks707

    Figure Lengend Snippet: ATM-mediated dCK Serine 74 phosphorylation is required for the IR-induced G2/M checkpoint. ( A ) dCK knock-down HeLa cells were re-introduced with vector control, dCK wild-type, dCK Serine 74 to alanine (S74A) mutation or Serine 74 to Glutamic Acid (S74E) mutation. The complementation effects using RT–PCR and western blot are shown. ( B ) The dCK complemented cell lines were treated with mock or IR (6 Gy). Two hours after treatment, cells were harvested and stained for Histone H3 Ser10 phosphorylation followed by flow cytometry. ( C ) Expression of flag-tagged dCK as compared to endogenous dCK in control shRNA and ATM shRNA cells. ( D ) HeLa cells with control or ATM shRNA were transiently transfected with vector, wild-type (WT), S74A or S74E of dCK and treated with IR (6 Gy). Two hours after treatment, cells were harvested and stained for Histone H3 Ser10 phosphorylation followed by flow cytometry analysis. The average and standard deviations of at least triplicate samples are shown. Statistic analysis was done by Student’s t -test; * P ≤ 0.05.

    Article Snippet: In vitro kinase assay For the Cdk1 in vitro kinase assay, recombinant dCK (Novus Biologicals, Littleton, CO, USA) and Cdk1/CyclinA2 (Promega, Madison, WI, USA) were incubated with 1 μg of Histone H1 (Promega) in the presence of 50 µM ATP in kinase buffer for 30 min at room temperature.

    Techniques: Plasmid Preparation, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Flow Cytometry, Cytometry, Expressing, shRNA, Transfection

    dCK is required for the G2/M checkpoint in response to ionizing radiation. ( A ) Establishment of isogenic cell lines with stable dCK knock-down. HeLa cells were stably transfected with control or dCK shRNA. Individual clones were obtained under puromycin selection. Knock-down effects were confirmed by RT–PCR (left panel) and western blot (right panel). ( B ) The isogenic cell lines with control or dCK knock-down were treated with Ara-C at indicated doses for 24 h, and clonogenic survival was assessed. ( C ) Radiosensitivity was assessed by the colony formation assay in the isogenic cell lines after radiation treatment (0–6 Gy). ( D ) Isogenic HeLa cells were treated with mock or IR treatment (6 Gy). Two hours after treatment, cells were harvested and fixed followed by staining for Histone H3 Ser10 phosphorylation for flow cytometry. The average and standard deviations of at least triplicate samples are shown. Statistic analysis was done by Student’s t -test.

    Journal: Nucleic Acids Research

    Article Title: Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage

    doi: 10.1093/nar/gks707

    Figure Lengend Snippet: dCK is required for the G2/M checkpoint in response to ionizing radiation. ( A ) Establishment of isogenic cell lines with stable dCK knock-down. HeLa cells were stably transfected with control or dCK shRNA. Individual clones were obtained under puromycin selection. Knock-down effects were confirmed by RT–PCR (left panel) and western blot (right panel). ( B ) The isogenic cell lines with control or dCK knock-down were treated with Ara-C at indicated doses for 24 h, and clonogenic survival was assessed. ( C ) Radiosensitivity was assessed by the colony formation assay in the isogenic cell lines after radiation treatment (0–6 Gy). ( D ) Isogenic HeLa cells were treated with mock or IR treatment (6 Gy). Two hours after treatment, cells were harvested and fixed followed by staining for Histone H3 Ser10 phosphorylation for flow cytometry. The average and standard deviations of at least triplicate samples are shown. Statistic analysis was done by Student’s t -test.

    Article Snippet: In vitro kinase assay For the Cdk1 in vitro kinase assay, recombinant dCK (Novus Biologicals, Littleton, CO, USA) and Cdk1/CyclinA2 (Promega, Madison, WI, USA) were incubated with 1 μg of Histone H1 (Promega) in the presence of 50 µM ATP in kinase buffer for 30 min at room temperature.

    Techniques: Stable Transfection, Transfection, shRNA, Clone Assay, Selection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Acetylene Reduction Assay, Colony Assay, Staining, Flow Cytometry, Cytometry

    Western blot analysis of GR, p-CREB and Sp1 in liver nuclear protein extracts of LW and EHL pigets. Histone H1 was used as a loading control. The nuclear contents of the transcription factors are presented as the fold change relative to LW. Blank bars represent Large White (LW) piglets; filled bars represent Erhualian (EHL) piglets. Values are presented as mean ± SEM. ** donates P

    Journal: PLoS ONE

    Article Title: Breed-Dependent Transcriptional Regulation of 5?-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

    doi: 10.1371/journal.pone.0040432

    Figure Lengend Snippet: Western blot analysis of GR, p-CREB and Sp1 in liver nuclear protein extracts of LW and EHL pigets. Histone H1 was used as a loading control. The nuclear contents of the transcription factors are presented as the fold change relative to LW. Blank bars represent Large White (LW) piglets; filled bars represent Erhualian (EHL) piglets. Values are presented as mean ± SEM. ** donates P

    Article Snippet: Histone H1 (BS1656, Bioworld Technology, MN, USA) was used as a loading control.

    Techniques: Western Blot

    β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and histone H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.

    Journal: Biochemical and biophysical research communications

    Article Title: Nuclear AXIN2 represses MYC gene expression

    doi: 10.1016/j.bbrc.2013.11.089

    Figure Lengend Snippet: β-Catenin bridges AXIN2 to TCF4. (A) Western blot analysis of proteins prepared from whole cell [W], cytoplasmic [C], and nuclear [N] compartments of HCT116 cells expressing NLS-AXIN2. (B) Top , Co-immunoprecipitation/western blot analysis of proteins precipitated with anti-AXIN2 antibodies in nuclear lysates prepared from HCT116 cells. Bottom , Western blot analysis of cytoplasmic [C] and nuclear [N] fractions using α-tubulin and histone H3 antibodies. (C) Co-immunoprecipitation/western blot analysis of TCF4 interacting proteins. HEK293 cells were transfected with plasmids expressing the indicated cDNAs and TCF4 was precipitated with anti-TCF4 antibodies.

    Article Snippet: Each lane of an 8% polyacrylamide gel contained 20 μg of protein lysates and the blots were probed with the following primary antibodies: anti-AXIN2 (Santa Cruz, sc-20784, 1:250), anti-α-tubulin (Sigma, T9026, 1:1000), anti-histone H3 (Millipore, 07-690, 1:25000), anti-TCF4 (Millipore, 05-511, 1:1000), and anti-β-catenin (BD Transduction, 610154, 1:1000).

    Techniques: Western Blot, Expressing, Immunoprecipitation, Transfection

    Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .

    Journal: Cancer cell

    Article Title: Mutant IDH1 downregulates ATM and alters DNA repair and sensitivity to DNA damage independent of TET2

    doi: 10.1016/j.ccell.2016.05.018

    Figure Lengend Snippet: Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .

    Article Snippet: Primary antibodies (Abs) used in this study recognized: ATM (Genetex, Cat.No: GTX70103 or Santa Cruz, Cat.No: sc-23921), phospho-ATM (Rockland, Cat.No: 200–301–400), Chek2 (Millipore, Cat.No: 05–649), phospho-p53 (Ser18) (Cell Signaling Technology, Cat. No: 9284), p53 (Vector Laboratories, Cat. No: VP-P956), p21 (BD Pharmingen, Cat.No: 556431), β-actin (Sigma; Cat.No: A2066), histone H3 (Abcam, Cat.No: ab10799), histone H3 (trimethyl K9) (Abcam, Cat.No: ab8898), histone H3 (dimethyl K9) (Abcam, Cat.No: ab1220), histone H3 (trimethyl K27) (Millipore, Cat.No: 07–449), histone H3 (dimethyl K79) (Cell Signalling Technology, Cat.No: 9757), and histone H3 (trimethyl K36) (Abcam, Cat.No: ab9050).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Mouse Assay, Chromatin Immunoprecipitation, Isolation, Cell Culture, Quantitative RT-PCR

    Amino acid sequence and phosphorylated sites of bovine histone H1.2 as determined by mass spectrometry. Lysine residues are marked red, and those identified as targets for the phosphorylation by phosphoramidate (i.e. seven residues) are highlighted. Out of the 305 peptides that were used to identify the protein, 14 peptides were reported to contain phospholysine.

    Journal: Upsala Journal of Medical Sciences

    Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

    doi: 10.3109/03009734.2014.996720

    Figure Lengend Snippet: Amino acid sequence and phosphorylated sites of bovine histone H1.2 as determined by mass spectrometry. Lysine residues are marked red, and those identified as targets for the phosphorylation by phosphoramidate (i.e. seven residues) are highlighted. Out of the 305 peptides that were used to identify the protein, 14 peptides were reported to contain phospholysine.

    Article Snippet: Compared to this high number of lysine residues, the phosphorylation of histone H1 is quite low.

    Techniques: Sequencing, Mass Spectrometry

    Decrease in phosphate in phosphoramidate phosphorylated histone H1 (▪) and 30 kDa polylysine (⧫) during incubation with PHPT1. The concentration was 1 mg/mL of phosphohistone and 2 mg/mL of phosphopolylysine. An amount of 5 pmol PHPT1 was added per 51 µL incubation volume. The reaction was performed at pH 7.5 and 30°C during indicated times and was interrupted by centrifugation of 50 µL of the reaction mixture through a spin column containing 200 µL of DEAE-Sepharose equilibrated in 25 mM Tris/HCl pH 8.5. The protein-bound, acid-labile phosphate in the final eluate was analyzed as described under Methods. Each time point was analyzed in duplicate.

    Journal: Upsala Journal of Medical Sciences

    Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

    doi: 10.3109/03009734.2014.996720

    Figure Lengend Snippet: Decrease in phosphate in phosphoramidate phosphorylated histone H1 (▪) and 30 kDa polylysine (⧫) during incubation with PHPT1. The concentration was 1 mg/mL of phosphohistone and 2 mg/mL of phosphopolylysine. An amount of 5 pmol PHPT1 was added per 51 µL incubation volume. The reaction was performed at pH 7.5 and 30°C during indicated times and was interrupted by centrifugation of 50 µL of the reaction mixture through a spin column containing 200 µL of DEAE-Sepharose equilibrated in 25 mM Tris/HCl pH 8.5. The protein-bound, acid-labile phosphate in the final eluate was analyzed as described under Methods. Each time point was analyzed in duplicate.

    Article Snippet: Compared to this high number of lysine residues, the phosphorylation of histone H1 is quite low.

    Techniques: Incubation, Concentration Assay, Centrifugation

    MS/MS spectrum showing the fragmentation pattern of one of the peptides obtained after trypsin treatment of histone H1 from SignalChem. The y-ion series is shown in red, and the b-ions series is in blue. Also shown is y- and b-ions fitting with the loss of 18 Da in violet and turquoise, respectively. Other observed but unspecified mached ions are grey. The small inset at the top shows only the y- and b-ions, with the length of the staples representing the intensity of the ions.

    Journal: Upsala Journal of Medical Sciences

    Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

    doi: 10.3109/03009734.2014.996720

    Figure Lengend Snippet: MS/MS spectrum showing the fragmentation pattern of one of the peptides obtained after trypsin treatment of histone H1 from SignalChem. The y-ion series is shown in red, and the b-ions series is in blue. Also shown is y- and b-ions fitting with the loss of 18 Da in violet and turquoise, respectively. Other observed but unspecified mached ions are grey. The small inset at the top shows only the y- and b-ions, with the length of the staples representing the intensity of the ions.

    Article Snippet: Compared to this high number of lysine residues, the phosphorylation of histone H1 is quite low.

    Techniques: Mass Spectrometry

    A typical response of ENaC to exogenous PI(3,4,5)P 3 . Shown is a representative current trace from an excised, outside-out patch (V p = 0 mV) formed from a CHO cell expressing wild-type ENaC before and after addition of 20 μM exogenous diC8 PI(3,4,5)P 3 . PI(3,4,5)P 3 added to the bathing solution in the presence of histone H1 carrier. Amiloride subsequently added to the bath solution toward the end of the experiment. This representative patch contains, at least, five ENaC. Shown at top is a continuous trace. Shown below (left) at an expanded timescale are regions of the trace before (1. control; middle) and after (2. PIP 3 ; bottom) addition of exogenous phosphoinositide. Respective all-point histograms for the regions shown at an expanded time-scale are to the right. All other conditions the same as Fig. 3 A .

    Journal: The Journal of General Physiology

    Article Title: Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel

    doi: 10.1085/jgp.200709800

    Figure Lengend Snippet: A typical response of ENaC to exogenous PI(3,4,5)P 3 . Shown is a representative current trace from an excised, outside-out patch (V p = 0 mV) formed from a CHO cell expressing wild-type ENaC before and after addition of 20 μM exogenous diC8 PI(3,4,5)P 3 . PI(3,4,5)P 3 added to the bathing solution in the presence of histone H1 carrier. Amiloride subsequently added to the bath solution toward the end of the experiment. This representative patch contains, at least, five ENaC. Shown at top is a continuous trace. Shown below (left) at an expanded timescale are regions of the trace before (1. control; middle) and after (2. PIP 3 ; bottom) addition of exogenous phosphoinositide. Respective all-point histograms for the regions shown at an expanded time-scale are to the right. All other conditions the same as Fig. 3 A .

    Article Snippet: Stock phosphoinositides were mixed just before use with an equal volume of a carrier solution containing histone H1 (0.2 mM; Echelon Biosciences Inc.) and sonicated again for 10 min. Phosphoinositide plus carrier was added to the bathing solution by direct pippetting close to the patched membrane.

    Techniques: Expressing

    Effect of the expression of UL23 on the distribution of Nmi and STAT1 in nuclear and cytoplasmic fractions. (A) U251 (lanes 2 and 4) and U251-UL23 cells (lanes 1 and 3) were treated with IFN-γ (1000 U/ml) and harvested at 36 hours post-treatment. The IFN-γ treated U251 cells were infected with HCMV Towne BAC  (lanes 5 and 7) or ΔUL23 (lanes 6 and 8) (MOI = 1) at 12 hours post-treatment and harvested at 24 hours postinfection. The harvested cells were separated into nuclear and cytoplasmic fractions. Equivalent amounts of each fraction were analyzed by immunoblotting with anti-UL23, anti-Nmi, and anti-STAT1. The purity of the nuclear and cytoplasmic fractions was assayed by immunoblotting with anti-histone H1 and anti-actin, respectively. The membranes were reacted with antibodies and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [  60 ,  64 ]. The protein levels of Nmi (B) and STAT1 (C) in the nuclei and cytoplasm of different cells that were mock-infected or infected with different viruses were quantified. The experiments were repeated three times. The standard deviation is indicated by the error bar.

    Journal: PLoS Pathogens

    Article Title: Human cytomegalovirus UL23 inhibits transcription of interferon-γ stimulated genes and blocks antiviral interferon-γ responses by interacting with human N-myc interactor protein

    doi: 10.1371/journal.ppat.1006867

    Figure Lengend Snippet: Effect of the expression of UL23 on the distribution of Nmi and STAT1 in nuclear and cytoplasmic fractions. (A) U251 (lanes 2 and 4) and U251-UL23 cells (lanes 1 and 3) were treated with IFN-γ (1000 U/ml) and harvested at 36 hours post-treatment. The IFN-γ treated U251 cells were infected with HCMV Towne BAC (lanes 5 and 7) or ΔUL23 (lanes 6 and 8) (MOI = 1) at 12 hours post-treatment and harvested at 24 hours postinfection. The harvested cells were separated into nuclear and cytoplasmic fractions. Equivalent amounts of each fraction were analyzed by immunoblotting with anti-UL23, anti-Nmi, and anti-STAT1. The purity of the nuclear and cytoplasmic fractions was assayed by immunoblotting with anti-histone H1 and anti-actin, respectively. The membranes were reacted with antibodies and quantitated with a STORM840 PhosphorImager (GE Healthcare) or a Gel Documentation Station (BioRad, Hercules, CA) [ 60 , 64 ]. The protein levels of Nmi (B) and STAT1 (C) in the nuclei and cytoplasm of different cells that were mock-infected or infected with different viruses were quantified. The experiments were repeated three times. The standard deviation is indicated by the error bar.

    Article Snippet: We used equal amounts of cytoplasmic and nuclear extracts for immunoblotting of STAT1, Nmi, actin, and histone H1 (Proteintech, Manchester, United Kingdom).

    Techniques: Expressing, Infection, BAC Assay, Standard Deviation

    Chromosome segregation in meiosis I is abortive in Gwl-inhibited oocytes. (A and B) Immature starfish oocytes were first injected with HiLyte 488–labeled tubulin (green) and Alexa 568–labeled histone H1 (magenta), and then injected with neutralizing anti-Gwl antibody (anti-Gwl) or control IgG (cont. IgG) along with ZZ-IBB (that can deliver cytoplasmically injected IgG into the nucleus) or uninjected (None). After 1-MeAde addition, live-cell images were obtained using a confocal microscope to monitor formation of the meiosis I spindle. Gwl-inhibited oocytes failed to properly segregate homologous chromosomes (A). This phenotype may be classified into three types, although they overlapped in some cases: lagging (#1, magenta), congressed (#2, yellow), or scattered (#3, blue) chromosomes along with frequently multipolar spindles (asterisks). However, further coinjection into immature oocytes with Arpp19 that had been in vitro thiophosphorylated by Gwl (pS106-Arpp19), but not with wild-type Arpp19 (wt-Arpp19), restored homologous chromosome segregation (B). Time after 1-MeAde addition is indicated in each frame. Arrow, chromosomes; arrowhead, the first polar body. Bars, 5 µm. (C) Quantification of chromosome segregation failure displayed in A and B. Each color corresponds to #1, #2, and #3 in A and B, respectively. Numbers of independent experiments (more than three females) are indicated at each bar.

    Journal: The Journal of Cell Biology

    Article Title: Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

    doi: 10.1083/jcb.201307160

    Figure Lengend Snippet: Chromosome segregation in meiosis I is abortive in Gwl-inhibited oocytes. (A and B) Immature starfish oocytes were first injected with HiLyte 488–labeled tubulin (green) and Alexa 568–labeled histone H1 (magenta), and then injected with neutralizing anti-Gwl antibody (anti-Gwl) or control IgG (cont. IgG) along with ZZ-IBB (that can deliver cytoplasmically injected IgG into the nucleus) or uninjected (None). After 1-MeAde addition, live-cell images were obtained using a confocal microscope to monitor formation of the meiosis I spindle. Gwl-inhibited oocytes failed to properly segregate homologous chromosomes (A). This phenotype may be classified into three types, although they overlapped in some cases: lagging (#1, magenta), congressed (#2, yellow), or scattered (#3, blue) chromosomes along with frequently multipolar spindles (asterisks). However, further coinjection into immature oocytes with Arpp19 that had been in vitro thiophosphorylated by Gwl (pS106-Arpp19), but not with wild-type Arpp19 (wt-Arpp19), restored homologous chromosome segregation (B). Time after 1-MeAde addition is indicated in each frame. Arrow, chromosomes; arrowhead, the first polar body. Bars, 5 µm. (C) Quantification of chromosome segregation failure displayed in A and B. Each color corresponds to #1, #2, and #3 in A and B, respectively. Numbers of independent experiments (more than three females) are indicated at each bar.

    Article Snippet: 1 µl of oocytes extracts were incubated for 30 min at 25°C in a final volume of 10 µl containing 0.3 mg/ml histone H1 (Boehringer Ingelheim) and 0.2 µl of γ-[32 P]ATP.

    Techniques: Injection, Labeling, Microscopy, In Vitro

    Arpp19 is phosphorylated on Ser69 by cyclin B–Cdk1 regardless of the presence or absence of Gwl. (A and B) Arpp19 becomes phosphorylated even in enucleated oocytes after 1-MeAde addition. Nucleated or enucleated oocytes were treated with 1-MeAde, and 40 min later (equivalent to metaphase of meiosis I) conventional (A) and phos-tag (B) SDS-PAGE followed by anti-Arpp19 immunoblots were performed. Entry into M phase was confirmed by loss of pTyr15 in Cdk1. (C) Gwl restores enucleation effects on the phosphorylation pattern of Arpp19. Enucleated immature oocytes were uninjected or injected with recombinant Gwl (recGwl; inactive, WT; control kinase-dead, KD) and then treated with 1-MeAde. Phos-tag analysis as in B was performed in C–F. (D) Inhibition of Cdk1 prevents Arpp19 phosphorylation in enucleated oocytes. Enucleated oocytes were treated with 1-MeAde in the presence of the Cdk inhibitor roscovitine (20 µM; Ros) or control DMSO. (E) Arpp19 becomes phosphorylated not only on the Gwl site, Ser106, but also on a possible Cdk1 site, Ser69, after entry into M phase in nucleated oocytes. Nucleated immature oocytes were injected with wild type (WT) or each mutant of Arpp19 proteins (4A [T42A, S69A, S106A, and T155A], 2A [S69A and S106A], and 3A [T42A, S106A, and T155A]), and then treated with 1-MeAde. The 3A mutant, which retains Ser69, showed the band upshift. (F) Cyclin B–Cdk1 directly phosphorylates Arpp19 on Ser69 in vitro. Each of wild-type Arpp19 and the mutant proteins described in E was mixed with purified cyclin B–Cdk1. Wild type and the 3A mutant, both of which retain Ser69, showed the band upshift. (G and H) Arpp19 is phosphorylated on Ser69 in vivo after entry into M phase both in nucleated and enucleated oocytes. Nucleated (G) or enucleated (H) oocytes were treated with 1-MeAde, and oocyte extracts were prepared at the indicated times. Anti-Arpp19 immunoprecipitates were analyzed with anti-pSer69 antibody. Entry into M phase was confirmed by loss of pTyr15 in Cdk1 and by histone H1 kinase (H1K) activation. Enucleation was confirmed by loss of MCM2, a nuclear protein marker (H). Brightness, contrast, and gamma settings were adjusted in the image presentation.

    Journal: The Journal of Cell Biology

    Article Title: Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

    doi: 10.1083/jcb.201307160

    Figure Lengend Snippet: Arpp19 is phosphorylated on Ser69 by cyclin B–Cdk1 regardless of the presence or absence of Gwl. (A and B) Arpp19 becomes phosphorylated even in enucleated oocytes after 1-MeAde addition. Nucleated or enucleated oocytes were treated with 1-MeAde, and 40 min later (equivalent to metaphase of meiosis I) conventional (A) and phos-tag (B) SDS-PAGE followed by anti-Arpp19 immunoblots were performed. Entry into M phase was confirmed by loss of pTyr15 in Cdk1. (C) Gwl restores enucleation effects on the phosphorylation pattern of Arpp19. Enucleated immature oocytes were uninjected or injected with recombinant Gwl (recGwl; inactive, WT; control kinase-dead, KD) and then treated with 1-MeAde. Phos-tag analysis as in B was performed in C–F. (D) Inhibition of Cdk1 prevents Arpp19 phosphorylation in enucleated oocytes. Enucleated oocytes were treated with 1-MeAde in the presence of the Cdk inhibitor roscovitine (20 µM; Ros) or control DMSO. (E) Arpp19 becomes phosphorylated not only on the Gwl site, Ser106, but also on a possible Cdk1 site, Ser69, after entry into M phase in nucleated oocytes. Nucleated immature oocytes were injected with wild type (WT) or each mutant of Arpp19 proteins (4A [T42A, S69A, S106A, and T155A], 2A [S69A and S106A], and 3A [T42A, S106A, and T155A]), and then treated with 1-MeAde. The 3A mutant, which retains Ser69, showed the band upshift. (F) Cyclin B–Cdk1 directly phosphorylates Arpp19 on Ser69 in vitro. Each of wild-type Arpp19 and the mutant proteins described in E was mixed with purified cyclin B–Cdk1. Wild type and the 3A mutant, both of which retain Ser69, showed the band upshift. (G and H) Arpp19 is phosphorylated on Ser69 in vivo after entry into M phase both in nucleated and enucleated oocytes. Nucleated (G) or enucleated (H) oocytes were treated with 1-MeAde, and oocyte extracts were prepared at the indicated times. Anti-Arpp19 immunoprecipitates were analyzed with anti-pSer69 antibody. Entry into M phase was confirmed by loss of pTyr15 in Cdk1 and by histone H1 kinase (H1K) activation. Enucleation was confirmed by loss of MCM2, a nuclear protein marker (H). Brightness, contrast, and gamma settings were adjusted in the image presentation.

    Article Snippet: 1 µl of oocytes extracts were incubated for 30 min at 25°C in a final volume of 10 µl containing 0.3 mg/ml histone H1 (Boehringer Ingelheim) and 0.2 µl of γ-[32 P]ATP.

    Techniques: SDS Page, Western Blot, Injection, Recombinant, Inhibition, Mutagenesis, In Vitro, Purification, In Vivo, Activation Assay, Marker

    Nuclear translocation of Nrf2 leads to drug resistance under hypoxia A-C. MCF7 cells were exposed to hypoxia for 0, 4, 8, and 24 hours. (A) Nrf2 localization was determined by immunocytochemistry (ICC) with an anti-Nrf2 antibody (green fluorescence). Nuclear location was determined by DAPI staining (blue fluorescence). (B, C) Total proteins and cytosolic (C)/nuclear (N) proteins were extracted. The protein levels of Hif-1α, Nrf2, GAPDH, and Histone H3 were detected by western blotting. GAPDH was the loading control for the cytosolic fraction, and Histone H3 was the loading control for the nuclear fraction. D. MCF7 cells were transfected with pARE-CMV and pRL-CMV plasmids. After transfection, the cells were exposed to hypoxia for 0, 4, 8, and 24 hours, and the firefly and Renilla luminescence activities were detected by Dual-Glo luciferase assay. The luciferase activity was represented by firefly luminescence normalized with Renilla luminescence. N=3, *, P

    Journal: Oncotarget

    Article Title: Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia

    doi: 10.18632/oncotarget.7406

    Figure Lengend Snippet: Nuclear translocation of Nrf2 leads to drug resistance under hypoxia A-C. MCF7 cells were exposed to hypoxia for 0, 4, 8, and 24 hours. (A) Nrf2 localization was determined by immunocytochemistry (ICC) with an anti-Nrf2 antibody (green fluorescence). Nuclear location was determined by DAPI staining (blue fluorescence). (B, C) Total proteins and cytosolic (C)/nuclear (N) proteins were extracted. The protein levels of Hif-1α, Nrf2, GAPDH, and Histone H3 were detected by western blotting. GAPDH was the loading control for the cytosolic fraction, and Histone H3 was the loading control for the nuclear fraction. D. MCF7 cells were transfected with pARE-CMV and pRL-CMV plasmids. After transfection, the cells were exposed to hypoxia for 0, 4, 8, and 24 hours, and the firefly and Renilla luminescence activities were detected by Dual-Glo luciferase assay. The luciferase activity was represented by firefly luminescence normalized with Renilla luminescence. N=3, *, P

    Article Snippet: Antibodies, chemicals, siRNA and plasmids Primary antibodies specific for HIF-1α, Nrf2, p-Nrf2, GCLM, MRP-1, GAPDH, and Histone H3 were purchased from Genetex.

    Techniques: Translocation Assay, Immunocytochemistry, Fluorescence, Staining, Western Blot, Transfection, Luciferase, Activity Assay

    ATPase-dependent activity of the Mms21 SUMO ligase. A . Growth test of GALp-SMC5 cells expressing wild-type SMC5 , smc5(K75I) , or smc5(D1014A) from a centromeric vector in plates containing galactose ( GALp ON) or glucose ( GALp OFF). B . Mms21-3HA was immunoprecipitated from exponentially growing cells transformed with the indicated SMC5 -expressing centromeric plasmids to test the Smc5-Mms21 interaction; wt = wild type; KI = smc5(K75I) ; DA = smc5(D1014A) . C . Sumoylation analysis of ATPase-defective Smc5-9myc proteins. HF-SUMO pull-down analysis in wild-type cells transformed with plasmids expressing the indicated SMC5 alleles. D . Sumoylation analysis of Nse4-6HA in smc5 ATPase mutant cells. HF-SUMO pull-down analysis in GALp-SMC5 NSE4-6HA cells expressing the indicated SMC5 alleles. Cells were shifted to glucose 6 h before collection to repress the endogenous SMC5 gene. E . Sumoylation analysis of cohesin in smc5 ATPase mutant cells. HF-SUMO pull down from cells of the indicated genotype (wt, mms21ΔC and GALp-SMC5 ), carrying a C-terminal 6HA tag on SMC1 , and expressing or not an ectopic copy of SMC5-9myc (WT) or smc5(K75I)-9myc (KI) allele; where indicated, cells were treated with MMS 0,02% for 1 h (MMS) before collection. F . Chromatin fractionation assay from GALp-SMC5 MMS21-6HA cells expressing an ectopic 9myc-tagged copy of the indicated SMC5 alleles, collected 6 h after shift to glucose to deplete the endogenous Smc5 protein. Controls for a chromatin-bound protein (histone H3), nuclear soluble (Rpd3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown; WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction. In C–E, arrow points to unmodified Smc5, Nse4, or Smc1 proteins, and vertical bars to their sumoylated forms.

    Journal: PLoS Biology

    Article Title: ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

    doi: 10.1371/journal.pbio.1002089

    Figure Lengend Snippet: ATPase-dependent activity of the Mms21 SUMO ligase. A . Growth test of GALp-SMC5 cells expressing wild-type SMC5 , smc5(K75I) , or smc5(D1014A) from a centromeric vector in plates containing galactose ( GALp ON) or glucose ( GALp OFF). B . Mms21-3HA was immunoprecipitated from exponentially growing cells transformed with the indicated SMC5 -expressing centromeric plasmids to test the Smc5-Mms21 interaction; wt = wild type; KI = smc5(K75I) ; DA = smc5(D1014A) . C . Sumoylation analysis of ATPase-defective Smc5-9myc proteins. HF-SUMO pull-down analysis in wild-type cells transformed with plasmids expressing the indicated SMC5 alleles. D . Sumoylation analysis of Nse4-6HA in smc5 ATPase mutant cells. HF-SUMO pull-down analysis in GALp-SMC5 NSE4-6HA cells expressing the indicated SMC5 alleles. Cells were shifted to glucose 6 h before collection to repress the endogenous SMC5 gene. E . Sumoylation analysis of cohesin in smc5 ATPase mutant cells. HF-SUMO pull down from cells of the indicated genotype (wt, mms21ΔC and GALp-SMC5 ), carrying a C-terminal 6HA tag on SMC1 , and expressing or not an ectopic copy of SMC5-9myc (WT) or smc5(K75I)-9myc (KI) allele; where indicated, cells were treated with MMS 0,02% for 1 h (MMS) before collection. F . Chromatin fractionation assay from GALp-SMC5 MMS21-6HA cells expressing an ectopic 9myc-tagged copy of the indicated SMC5 alleles, collected 6 h after shift to glucose to deplete the endogenous Smc5 protein. Controls for a chromatin-bound protein (histone H3), nuclear soluble (Rpd3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown; WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction. In C–E, arrow points to unmodified Smc5, Nse4, or Smc1 proteins, and vertical bars to their sumoylated forms.

    Article Snippet: All proteins were resolved in 10% SDS-PAGE gels, except SMC proteins (7.5%), histone H3 (15%), and (4%–15% gradient gel; BioRad).

    Techniques: Activity Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transformation Assay, Mutagenesis, Fractionation

    Mms21-dependent sumoylation requires an intact Smc5/6 complex. A . Composition of Smc5/6, depicting the different entities present in the complex. Nse subunits are labeled 1 to 6; Nse2 = Mms21. B . Sumoylation of Smc5 in smc6 mutant cells. Samples of wild type and GAL-3HA-SMC6 were collected from cells growing exponentially in galactose ( GALp ON), or 12 h after shift to glucose to repress 3HA-SMC6 expression ( GALp OFF). A GALp-3HA-SMC6 strain expressing the smc6-1 allele from a centromeric vector was also included in the analysis. Protein extracts were processed for HF-SUMO pull down as in Fig. 1G . C . Co-immunoprecipitation analysis of the Smc5-Mms21 interaction from wild type and smc6-1 protein extracts. Wild type and GALp-SMC6 smc6-1 cells expressing Smc5-9myc and Mms21-6HA were shifted to glucose for 12 h and processed for anti-HA immunoprecipitation. D . Chromatin fractionation assay from wild type and smc6-1 cells to analyze the amount of chromatin-bound Mms21-6HA. Controls for a chromatin-bound protein (histone H3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown. E . Temperature and methyl methanesulfonate (MMS)-sensitivity of nse hypomorphic alleles. Growth test of wild type, nse3-2 , and nse5-2 cells in YPD plates at 25°C (containing or not the indicated MMS concentration) or at 37°C. F . Analysis of the Smc5-Nse3 and Smc5-Nse5 interaction in nse hypomorphic alleles. Exponentially growing Smc5-6Flag cells, expressing 9myc-tagged versions of either the wild type or the indicated hypomorphic nse alleles, were shifted to 37°C for 2 h (37) or kept at 25°C (25) before Smc5-6Flag immunoprecipitation. G . Co-immunoprecipitation analysis of the Smc5-Mms21 interaction in nse3-2 and nse5-2 mutant cells. Smc5-6Flag was immunoprecipitated, as in F, from cells grown at the indicated temperatures. Co-immunoprecipitation of Mms21-6HA was analyzed by western blot. H . Chromatin fractionation assay from Mms21-6HA tagged wild type and nse5-2 cells, as in D. I . HF-SUMO pull down from wild type, nse3-2 , or nse5-2 cells expressing Smc5-6HA, before and after a shift to 37°C. In B and G, arrow points to unmodified form of the proteins, vertical bar to sumoylated forms. In D and H, WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction.

    Journal: PLoS Biology

    Article Title: ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair

    doi: 10.1371/journal.pbio.1002089

    Figure Lengend Snippet: Mms21-dependent sumoylation requires an intact Smc5/6 complex. A . Composition of Smc5/6, depicting the different entities present in the complex. Nse subunits are labeled 1 to 6; Nse2 = Mms21. B . Sumoylation of Smc5 in smc6 mutant cells. Samples of wild type and GAL-3HA-SMC6 were collected from cells growing exponentially in galactose ( GALp ON), or 12 h after shift to glucose to repress 3HA-SMC6 expression ( GALp OFF). A GALp-3HA-SMC6 strain expressing the smc6-1 allele from a centromeric vector was also included in the analysis. Protein extracts were processed for HF-SUMO pull down as in Fig. 1G . C . Co-immunoprecipitation analysis of the Smc5-Mms21 interaction from wild type and smc6-1 protein extracts. Wild type and GALp-SMC6 smc6-1 cells expressing Smc5-9myc and Mms21-6HA were shifted to glucose for 12 h and processed for anti-HA immunoprecipitation. D . Chromatin fractionation assay from wild type and smc6-1 cells to analyze the amount of chromatin-bound Mms21-6HA. Controls for a chromatin-bound protein (histone H3) and cytoplasmic soluble (Hexokinase; Hxk) proteins are shown. E . Temperature and methyl methanesulfonate (MMS)-sensitivity of nse hypomorphic alleles. Growth test of wild type, nse3-2 , and nse5-2 cells in YPD plates at 25°C (containing or not the indicated MMS concentration) or at 37°C. F . Analysis of the Smc5-Nse3 and Smc5-Nse5 interaction in nse hypomorphic alleles. Exponentially growing Smc5-6Flag cells, expressing 9myc-tagged versions of either the wild type or the indicated hypomorphic nse alleles, were shifted to 37°C for 2 h (37) or kept at 25°C (25) before Smc5-6Flag immunoprecipitation. G . Co-immunoprecipitation analysis of the Smc5-Mms21 interaction in nse3-2 and nse5-2 mutant cells. Smc5-6Flag was immunoprecipitated, as in F, from cells grown at the indicated temperatures. Co-immunoprecipitation of Mms21-6HA was analyzed by western blot. H . Chromatin fractionation assay from Mms21-6HA tagged wild type and nse5-2 cells, as in D. I . HF-SUMO pull down from wild type, nse3-2 , or nse5-2 cells expressing Smc5-6HA, before and after a shift to 37°C. In B and G, arrow points to unmodified form of the proteins, vertical bar to sumoylated forms. In D and H, WCE: Whole Cell Extract; SN: Supernatant; Chr: Chromatin fraction.

    Article Snippet: All proteins were resolved in 10% SDS-PAGE gels, except SMC proteins (7.5%), histone H3 (15%), and (4%–15% gradient gel; BioRad).

    Techniques: Labeling, Mutagenesis, Expressing, Plasmid Preparation, Immunoprecipitation, Fractionation, Concentration Assay, Western Blot