anti-h2b Search Results


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  • 98
    Millipore anti h2b
    The presence of NETs (MPO and histones: H2A, <t>H2B,</t> and H3) in the chorion and the placental decidua. Chromogen 3.3′-diaminobenzidine: (1) anti-MPO, (2) anti-H2A, (3) anti-H2B, (4) anti-H3 antibodies; ( A ) control group, without granulocytes (magnification 200x); ( B ) women with miscarriage, with granulocytes (magnification 400x); ( C ) women with miscarriage, with NETs (arrows) (magnification 400x).
    Anti H2b, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Abcam anti h2b
    Altered chromatin structure and/or human BRE1 complex recruitment to chromatin is crucial for efficient <t>H2B</t> ubiquitylation. (A) The chromatin ubiquitylation assay was performed with the indicated substrates for H2B ubiquitylation (top). Analyses of purified recombinant histone octamers and natural oligonucleosome derived from HeLa cells by SDS-PAGE with Coomassie Blue staining to ensure comparable levels of protein usage for in vitro chromatin ubiquitylation assay (bottom). Note that endogenous ubH2B is not detected in lane 2 because a much smaller amount (350 ng) of nucleosome substrate was used in this assay. (B) hBRE1C recruitment to chromatin is critical for efficient H2B ubiquitylation. A recombinant chromatin template containing the pG 5 ML plasmid was subjected to the in vitro ubiquitylation assay with hE1, hRAD6A, ubiquitin and either hBRE1C or GAL4-hBRE1C, as indicated, and in the continued presence of the chromatin assembly factors. Reaction products were analyzed by immunoblot with indicated antibodies.
    Anti H2b, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam histone h2b
    SMC 5 functions jointly with DDX 11 to promote DNA repair Left panel: Growth curve was carried out to test ddx11 smc5‐aid cell proliferation after SMC5‐AID depletion induced by auxin treatment. Data represent means ± SD of three experiments. Experiments were carried out at 39.5°C. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Tubulin was used as loading control. Survival curve of ddx11 smc5‐aid cells treated with different concentrations of cisplatin after SMC5 depletion upon auxin treatment. Data represent means ± SD of four experiments. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Histone <t>H2B</t> was used as loading control.
    Histone H2b, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 267 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti histone h2b
    Subcellular localization of stably expressed Ptc1. (A) Schematic representation of stably transfected full-length human Ptc1 protein with possess 12 transmembrane domains. Red box at N-terminus indicates 3×T7-tag, green box at end of intracellular domain indicates C-terminal 3×FLAG-tag, respectively. (B) Western blot analyses of Ptc1 protein stably expressed in HeLa cells. Ptc1 immunoprecipitated from cell extracts with C-terminal FLAG-tag and immunoblotted with anti-N-terminal T7 tag antibody (left panel) or anti-C-terminal FLAG tag antibody (right panel). Anti-T7 blot detects doublet bands corresponding to full-length Ptc1 (Ptc1 FL), while anti-FLAG blot reveals small C-terminal fragments representing the intracellular domain of Ptc1 (ICD7 fragments) as well as Ptc1 FL. (C) Multiple staining of Ptc1-stably expressing cells by anti-T7 tag immunostain (a: Ptc1 N-terminus), anti-FLAG-tag immunostain (b: Ptc1 C-terminus), DAPI DNA stain (c: nucleus) and their merged image (d). Scale bar, 20 µm. (D) Cell fractionation analysis of Ptc1-stably expressing cells. BIP and Histone <t>H2B</t> used as cytoplasmic and nuclear marker, respectively. Ptc1 FL (+), cells stably expressing full-length Ptc1; Ptc1 FL (−), control HeLa cells.
    Anti Histone H2b, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc histone h2b
    Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with <t>H2B</t> wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P
    Histone H2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc histone 2b
    P2X6 subunit interacts with SF3A1 and Spectrin α2 in nuclear extracts from hippocampus of 8 month-old mice and alters splicing efficiency  in vitro . ( a ) Western blot of subcellular fractions from 8 month-old mice hippocampus. P2X6 subunit was located in both the cytosolic (cyt) and the nuclear fraction (nuc), as identified by localization with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NeuN (neuronal nuclei marker)/histone 2b (H2b), respectively. ( b ) Nuclear extracts immunoprecipitated with anti-P2X6 antibody were resolved in a 2D electrophoresis gel and selected spots were isolated, processed and identified by MALDI/TOF (n = 3 mice). ( c-f ) Nuclear extracts immunoprecipitated with either IgG or anti-P2X6 antibodies were analysed by immunoblotting with antibodies against SF3A1 ( c ) and spectrin α-2( d ). Nuclear extracts were also immunoprecipitated with spectrin α-2 ( e ) or SF3A1 ( f ) antibodies and immunoblotted with anti-P2X6 antibody. ( g ) Scheme shows four DNA constructions prepared for splicing activity quantitation and their respective protein translations. ( h ) Splicing inhibitor isoginkgetin (IGK) was used to validate the molecular tools employed to quantify the splicing efficiency. As expected TPI I firefly luciferase-dependent luminescence decreases and TPI II firefly luciferase-dependent luminescence increases when splicing is impaired (mean±s.e.m., n = 5 independent experiments, * P
    Histone 2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti histone 2b
    Chl1 expression and chromatin binding are induced during S-phase. A) Logarithmically growing cells expressing Chl1-13Myc (YBS 1129) were harvested and analyzed for chromatin binding with or without MMS exposure. Immunoblots show whole cells extracts (WCE), cytoplasmic fractions (Cyt) and chromatin bound fractions (CB). <t>Histone</t> 2B (H2B) and Phosphoglycerate Kinase (PGK) were probed in parallel as positive controls for chromatin-bound and cytoplasmic proteins, respectively. B) Logarithmically growing cells expressing Chl1-13MYC were synchronized in G1 (alpha factor) and released into fresh medium. Samples were harvested every 15minutes and analyzed by Immunoblotting for Chl1-13MYC and Swi6 as a loading control. Parallel blots were also analyzed for Histone 2B (H2B) and Phosphoglyerinkinase (PGK) to confirm chromatin and cytoplasmic fractions, respectively. C) G1 synchronized cells expressing Chl1-13Myc were released in fresh medium and samples collected every 15 minutes, processed for chromatin binding and probed to detect Chl1-13MYC and Swi6 (loading control). D) Data shown are Chl1 protein levels relative to Swi6 (blue line) and chromatin enrichment relative to Swi6 (red line) over the cell cycle averaged from 3 independent experiments. Shaded portion denotes S-phase. E) Flow cytometric data for cells analyzed in B) and C).
    Anti Histone 2b, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti histone h2b
    Polyglutamine expansion of ATXN7 alters chromatin occupancy and histone <t>H2B</t> monoubiquitination at the RELN promoter and increases global H2B monoubiquitination. ( A ) Polyglutamine expansion of ATXN7 resulted in a reduction of polyQ ATXN7 occupancy at the
    Anti Histone H2b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Upstate Biotechnology Inc anti h2b
    Histones are on mammalian LDs and respond to LPS. ( A ). Western blot analysis of LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS. Antibodies against ALDI, histones H2A, <t>H2B</t> and H3 were used. Whole liver homogenate (h) was used for comparison and as a control. ( B ) The presence of histone H1 (H1) on LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS was detected by immunoblot, and more H1 was present on droplets purified from LPS-treated animals. Equal total proteins from the nuclear fraction (n) and from whole liver homogenate (h) were used as comparison. ( C ). Mice were injected intraperitoneally with (+) or without (−) LPS, and transaminase levels (AST and ALT) and cytokine levels (IL-6 and TNFα) were quantified in units/l or units/ml in the serum; asterisk indicates statistical significance (p=0.05), confirming that LPS injection provoked the expected biological response. ( D ). Western blot analysis of histone H1 released into the buffer (UB) when purified LDs, from the liver of infection induced mice, were treated with LPS (+). Histone H1 is either minimally detected or not at all detected in the buffer in the absence of LPS (−). The band at 97 kDa in C and D represents histone H1 oligomers. DOI: http://dx.doi.org/10.7554/eLife.00003.011
    Anti H2b, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti histone h2b antibody mabcam 52484 chip grade
    Histones are on mammalian LDs and respond to LPS. ( A ). Western blot analysis of LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS. Antibodies against ALDI, histones H2A, <t>H2B</t> and H3 were used. Whole liver homogenate (h) was used for comparison and as a control. ( B ) The presence of histone H1 (H1) on LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS was detected by immunoblot, and more H1 was present on droplets purified from LPS-treated animals. Equal total proteins from the nuclear fraction (n) and from whole liver homogenate (h) were used as comparison. ( C ). Mice were injected intraperitoneally with (+) or without (−) LPS, and transaminase levels (AST and ALT) and cytokine levels (IL-6 and TNFα) were quantified in units/l or units/ml in the serum; asterisk indicates statistical significance (p=0.05), confirming that LPS injection provoked the expected biological response. ( D ). Western blot analysis of histone H1 released into the buffer (UB) when purified LDs, from the liver of infection induced mice, were treated with LPS (+). Histone H1 is either minimally detected or not at all detected in the buffer in the absence of LPS (−). The band at 97 kDa in C and D represents histone H1 oligomers. DOI: http://dx.doi.org/10.7554/eLife.00003.011
    Anti Histone H2b Antibody Mabcam 52484 Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam anti histone h2b antibody
    Representative images, size distribution and fluorescence signal intensity from fluorescence marker of canonical histones (H2A, <t>H2B,</t> H3, H4) and 2 large variants of histone H2A (macroH2A1.1 and macroH2A1.2). ImageStream photographs show bright-field images and histone staining (fluorescence from Alexa Fluor® 488, Alexa Fluor® 549, Alexa Fluor® 594 or Alexa Fluor® 647)
    Anti Histone H2b Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Epitomics anti histone 2b
    MK-4 inhibits rotenone-induced nuclear accumulation of the NF-κB p65 subunit in BV2 cells. (A) Various doses of MK-4 and 1 μmol/L rotenone were administered to BV2 cells as indicated for 6 h. The cells were fixed and labeled with anti-p65 (red) antibodies. Then, the cells were visualized under a fluorescent microscope. The nuclei were stained with DAPI (1 μg/mL) (blue). Scale bars, 5 μm. (B) The cytoplasmic and nuclear fractions from the treated BV2 cells were immunoblotted with anti-p65, anti-GAPDH or anti-histone 2B antibodies. The band intensities of p65 relative to that of GAPDH (cytoplasmic fraction) or <t>histone</t> 2B (nuclear fraction) are shown in the lower two panels. The value of the group without drug (dissolvent only) is normalized as 1. The data are presented as the mean±SEM from three independent experiments. * P
    Anti Histone 2b, supplied by Epitomics, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA anti h2b
    Histones lost from LPS-challenged BMDMs are released into the medium, in soluble and EV-associated forms. (A) The supernatant of BMDMs challenged with LPS for 4 h was collected, centrifuged to eliminate cell debris, and then large vesicles that were retrieved separately [macrobodies (MBs)]. The concentrated cell-conditioned medium (CCM) was then loaded onto a discontinuous Optiprep density gradient. Twelve fractions were collected, and an aliquot was tested by Western blotting for the presence of histone H3; the remainder was centrifuged, and the pellets were dissolved in hot SDS-PAGE loading buffer. Microvesicles and exosomes (MEs)-containing fractions were identified by the presence of TSG101, a membrane marker ( 19 ). One representative experiment, of four performed, is shown. (B) Extracellular vesicles in supernatants of <t>H2B-GFP-expressing</t> BMDMs were trapped by beads coated with anti-CD63 antibodies; beads were analyzed for GFP fluorescence by flow cytometry. MEs contain distinctly more H2B-GFP than macrobodies ( n = 3, t -test, * p
    Anti H2b, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti p histone h2b s32
    Impairment of Aurora-B-mediated activities on <t>H2B</t> and HIPK2 induces cytokinesis failure. a , b HeLa cells were transfected with vectors carrying the indicated GFP-HIPK2 forms and stained 24 h post-transfection with <t>anti-p-Histone</t> H2B-S14 and Hoechst. The percentages of midbodies positive for H2B-S14 P staining in the transfected populations a and the percentages of binucleated cells also in the transfected populations b are reported as mean ± SD of two different experiments. c HF were transfected with vectors carrying GFP-tagged H2B-WT or the non-phosphorylatable H2B-S32A derivative. Cells were stained with anti-β-tubulin Ab and Hoechst. The percentages of binucleated cells in the GFP-positive populations were evaluated at the indicated time-points post-transfection and reported as fold change relative to that of GFP-H2B-WT in two different experiments. * p
    Anti P Histone H2b S32, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The presence of NETs (MPO and histones: H2A, H2B, and H3) in the chorion and the placental decidua. Chromogen 3.3′-diaminobenzidine: (1) anti-MPO, (2) anti-H2A, (3) anti-H2B, (4) anti-H3 antibodies; ( A ) control group, without granulocytes (magnification 200x); ( B ) women with miscarriage, with granulocytes (magnification 400x); ( C ) women with miscarriage, with NETs (arrows) (magnification 400x).

    Journal: Scientific Reports

    Article Title: Biomarkers of neutrophil extracellular traps (NETs) and nitric oxide-(NO)-dependent oxidative stress in women who miscarried

    doi: 10.1038/s41598-020-70106-x

    Figure Lengend Snippet: The presence of NETs (MPO and histones: H2A, H2B, and H3) in the chorion and the placental decidua. Chromogen 3.3′-diaminobenzidine: (1) anti-MPO, (2) anti-H2A, (3) anti-H2B, (4) anti-H3 antibodies; ( A ) control group, without granulocytes (magnification 200x); ( B ) women with miscarriage, with granulocytes (magnification 400x); ( C ) women with miscarriage, with NETs (arrows) (magnification 400x).

    Article Snippet: Nuclear chromatin was imaged using anti-H2A, anti-H2B, and anti-H3 antibodies.

    Techniques:

    Proposed mechanism of NO/NETs-dependent oxidative stress formation in women who had miscarried. NOS-dependent neutrophils-derived NO promotes miscarriage by lipid peroxidation, by NT and MDA. NOX-dependent stimulating ROS production, could affect miscarriage. We hypothesized if NOX-dependent NETs forming is the cause of miscarriage. Based on observed two population of measured placenta and serum markers we suggest two different NETs-involving mechanisms occurrences of miscarriage. In NETs positive patients (increase MPO and PTX3, decrease NO and cfDNA in serum), miscarriages were supported by direct harmful influence of NETs components. Whereas, in NETs negative patients (increase NO and NT, decrease PADI4 and cfDNA, but increase MPO and PTX3 in serum) with very high-level of oxidative stress markers (NO and NT)—dysregulation of oxidative and antioxidant balance, which suggest oxidative stress-dependent impairment and miscarriage. Abbreviations: NETs, neutrophil extracellular traps; NOS, nitric oxide synthase; NOX, NADPH oxidase; ROS, reactive oxygen species; NT, nitrotyrosine; MDA, malondialdehyde; PADI4, peptidylarginine deiminase 4; cfDNA, cyrkulating free DNA; MPO, myeloperoxidase; PTX-3, pentraxin 3; histones 2A, 2B, 3—H2A, H2B, H3.

    Journal: Scientific Reports

    Article Title: Biomarkers of neutrophil extracellular traps (NETs) and nitric oxide-(NO)-dependent oxidative stress in women who miscarried

    doi: 10.1038/s41598-020-70106-x

    Figure Lengend Snippet: Proposed mechanism of NO/NETs-dependent oxidative stress formation in women who had miscarried. NOS-dependent neutrophils-derived NO promotes miscarriage by lipid peroxidation, by NT and MDA. NOX-dependent stimulating ROS production, could affect miscarriage. We hypothesized if NOX-dependent NETs forming is the cause of miscarriage. Based on observed two population of measured placenta and serum markers we suggest two different NETs-involving mechanisms occurrences of miscarriage. In NETs positive patients (increase MPO and PTX3, decrease NO and cfDNA in serum), miscarriages were supported by direct harmful influence of NETs components. Whereas, in NETs negative patients (increase NO and NT, decrease PADI4 and cfDNA, but increase MPO and PTX3 in serum) with very high-level of oxidative stress markers (NO and NT)—dysregulation of oxidative and antioxidant balance, which suggest oxidative stress-dependent impairment and miscarriage. Abbreviations: NETs, neutrophil extracellular traps; NOS, nitric oxide synthase; NOX, NADPH oxidase; ROS, reactive oxygen species; NT, nitrotyrosine; MDA, malondialdehyde; PADI4, peptidylarginine deiminase 4; cfDNA, cyrkulating free DNA; MPO, myeloperoxidase; PTX-3, pentraxin 3; histones 2A, 2B, 3—H2A, H2B, H3.

    Article Snippet: Nuclear chromatin was imaged using anti-H2A, anti-H2B, and anti-H3 antibodies.

    Techniques: Derivative Assay, Multiple Displacement Amplification

    Altered chromatin structure and/or human BRE1 complex recruitment to chromatin is crucial for efficient H2B ubiquitylation. (A) The chromatin ubiquitylation assay was performed with the indicated substrates for H2B ubiquitylation (top). Analyses of purified recombinant histone octamers and natural oligonucleosome derived from HeLa cells by SDS-PAGE with Coomassie Blue staining to ensure comparable levels of protein usage for in vitro chromatin ubiquitylation assay (bottom). Note that endogenous ubH2B is not detected in lane 2 because a much smaller amount (350 ng) of nucleosome substrate was used in this assay. (B) hBRE1C recruitment to chromatin is critical for efficient H2B ubiquitylation. A recombinant chromatin template containing the pG 5 ML plasmid was subjected to the in vitro ubiquitylation assay with hE1, hRAD6A, ubiquitin and either hBRE1C or GAL4-hBRE1C, as indicated, and in the continued presence of the chromatin assembly factors. Reaction products were analyzed by immunoblot with indicated antibodies.

    Journal: Methods (San Diego, Calif.)

    Article Title: Nucleosomal H2B Ubiquitylation with Purified Factors

    doi: 10.1016/j.ymeth.2011.03.009

    Figure Lengend Snippet: Altered chromatin structure and/or human BRE1 complex recruitment to chromatin is crucial for efficient H2B ubiquitylation. (A) The chromatin ubiquitylation assay was performed with the indicated substrates for H2B ubiquitylation (top). Analyses of purified recombinant histone octamers and natural oligonucleosome derived from HeLa cells by SDS-PAGE with Coomassie Blue staining to ensure comparable levels of protein usage for in vitro chromatin ubiquitylation assay (bottom). Note that endogenous ubH2B is not detected in lane 2 because a much smaller amount (350 ng) of nucleosome substrate was used in this assay. (B) hBRE1C recruitment to chromatin is critical for efficient H2B ubiquitylation. A recombinant chromatin template containing the pG 5 ML plasmid was subjected to the in vitro ubiquitylation assay with hE1, hRAD6A, ubiquitin and either hBRE1C or GAL4-hBRE1C, as indicated, and in the continued presence of the chromatin assembly factors. Reaction products were analyzed by immunoblot with indicated antibodies.

    Article Snippet: The following antibodies are obtained commercially: anti-H2A (Millipore 07-146), anti-H2B (Abcam ab1790), anti-H3 (Abcam ab1791), anti-H4 (Abcam ab7311), anti-HA (Abcam ab9110), and anti-FLAG (HRP-conjugated, Sigma A8592).

    Techniques: Ubiquitin Assay, Purification, Recombinant, Derivative Assay, SDS Page, Staining, In Vitro, Plasmid Preparation

    Ctr2 −/−  BMMCs exhibit increased chymase and tryptase activity. (A) Cytospin slides were prepared from WT and Ctr2 −/−  BMMCs and stained for tryptase activity as described in Material and Methods. BMMCs isolated from Mcpt6 −/−  mice served as negative control for the tryptase activity staining. (B) Quantification of tryptase activity staining in WT and Ctr2 −/−  BMMCs. (C) Protein extracts isolated from WT and Ctr2 −/−  BMMCs were assayed for total trypsin-like activity using S-2288, total CPA activity using M-2245, and total chymase-like activity using a fluorescent substrate (Suc-Ala-Ala-Pro-Phe-AMC). Chymase-like activity is shown as raw fluorescent units (RFU). Total activities are given as activity per hour and 10 6  cells. (D) Protein extracts from WT and Ctr2 −/−  BMMCs were immunoblotted with anti-mMCP6, anti-CPA3, anti-mMCP4, anti-mMCP5, with anti-actin antibody as loading control. (E) Total mRNA extracts were prepared from WT and Ctr2 −/−  BMMCs and expression of Mcpt6, Cpa3, Mcpt1, Mcpt4, and Mcpt5 were analyzed with qPCR. (F) Microphthalmia transcription factor (Mitf), GATA1, GATA2, and PU.1 expression was analyzed as in (F). (G) Isolated proteins from WT and Ctr2 −/−  BMMCs were immunoblotted with anti-histone 3, anti-histone 2B, anti-mMCP6, and anti-actin antibody as loading control. Data are presented as mean ± S.E.M. from three - four biological replicates, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Ctr2 regulates mast cell maturation by affecting the storage and expression of tryptase and proteoglycans

    doi: 10.4049/jimmunol.1500283

    Figure Lengend Snippet: Ctr2 −/− BMMCs exhibit increased chymase and tryptase activity. (A) Cytospin slides were prepared from WT and Ctr2 −/− BMMCs and stained for tryptase activity as described in Material and Methods. BMMCs isolated from Mcpt6 −/− mice served as negative control for the tryptase activity staining. (B) Quantification of tryptase activity staining in WT and Ctr2 −/− BMMCs. (C) Protein extracts isolated from WT and Ctr2 −/− BMMCs were assayed for total trypsin-like activity using S-2288, total CPA activity using M-2245, and total chymase-like activity using a fluorescent substrate (Suc-Ala-Ala-Pro-Phe-AMC). Chymase-like activity is shown as raw fluorescent units (RFU). Total activities are given as activity per hour and 10 6 cells. (D) Protein extracts from WT and Ctr2 −/− BMMCs were immunoblotted with anti-mMCP6, anti-CPA3, anti-mMCP4, anti-mMCP5, with anti-actin antibody as loading control. (E) Total mRNA extracts were prepared from WT and Ctr2 −/− BMMCs and expression of Mcpt6, Cpa3, Mcpt1, Mcpt4, and Mcpt5 were analyzed with qPCR. (F) Microphthalmia transcription factor (Mitf), GATA1, GATA2, and PU.1 expression was analyzed as in (F). (G) Isolated proteins from WT and Ctr2 −/− BMMCs were immunoblotted with anti-histone 3, anti-histone 2B, anti-mMCP6, and anti-actin antibody as loading control. Data are presented as mean ± S.E.M. from three - four biological replicates, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

    Article Snippet: Primary antibodies used were against mouse MC protease 6 (mMCP6) (1:1000), CPA3 (1:500), mMCP4 (1:500), mMCP5 (1:500) (raised in rabbit as previously described , Histone 3 (1:2500), Histone 2B (1:2500) (Abcam).

    Techniques: Activity Assay, Staining, Isolation, Mouse Assay, Negative Control, Expressing, Real-time Polymerase Chain Reaction

    MN and nExo contain similar protein content. ( A ) Representative images of nuclear fraction and MN-enriched fraction. All samples were obtained from OVCAR-5 cells and stained with DAPI. The white box in the middle image is a magnified view. Scale bar, 50 μm. ( B ) Counts of protein cellular compartment of origin resulting from MS analysis in exosome and MN-enriched fraction. Samples were obtained from OVCAR-5 cells. The x axis represents the categories of cell components. Proteins from nuclear and chromosome compartments highlighted in yellow. ( C ) Venn diagram showing overlapping proteins between exosome and MN-enriched fraction. Samples were obtained from OVCAR-5 cells. Lamin A/C, Histone H2A/B, importin, and heat shock protein (HSP) 70/90 were included in the overlapped 127 proteins. ( D and E ) Serial confocal images of mCherry-LaminA/C–expressing OVCAR-5 cells. Nuclei were stained with DAPI. CD63 and CD9 were IF-stained with antibodies as described in Materials and Methods. Scale bars, 5 μm. ( F ) Representative images from time-lapse imaging in mCherry-LaminA/C–expressing OVCAR-5 cells. Cell membrane was stained with CellMask Deep Red. Cells stably expressed green fluorescent protein (GFP)–CD63 by lentiviral infections as described in Materials and Methods. Scale bar, 3 μm. ( G ) Representative images of time-lapse imaging in mCherry-Histone H2B–expressing OVCAR-5 cells. Cells were transiently transfected with mEmerald-CD9 plasmid as described in Materials and Methods. Scale bar, 4 μm.

    Journal: Science Advances

    Article Title: Mechanisms of nuclear content loading to exosomes

    doi: 10.1126/sciadv.aax8849

    Figure Lengend Snippet: MN and nExo contain similar protein content. ( A ) Representative images of nuclear fraction and MN-enriched fraction. All samples were obtained from OVCAR-5 cells and stained with DAPI. The white box in the middle image is a magnified view. Scale bar, 50 μm. ( B ) Counts of protein cellular compartment of origin resulting from MS analysis in exosome and MN-enriched fraction. Samples were obtained from OVCAR-5 cells. The x axis represents the categories of cell components. Proteins from nuclear and chromosome compartments highlighted in yellow. ( C ) Venn diagram showing overlapping proteins between exosome and MN-enriched fraction. Samples were obtained from OVCAR-5 cells. Lamin A/C, Histone H2A/B, importin, and heat shock protein (HSP) 70/90 were included in the overlapped 127 proteins. ( D and E ) Serial confocal images of mCherry-LaminA/C–expressing OVCAR-5 cells. Nuclei were stained with DAPI. CD63 and CD9 were IF-stained with antibodies as described in Materials and Methods. Scale bars, 5 μm. ( F ) Representative images from time-lapse imaging in mCherry-LaminA/C–expressing OVCAR-5 cells. Cell membrane was stained with CellMask Deep Red. Cells stably expressed green fluorescent protein (GFP)–CD63 by lentiviral infections as described in Materials and Methods. Scale bar, 3 μm. ( G ) Representative images of time-lapse imaging in mCherry-Histone H2B–expressing OVCAR-5 cells. Cells were transiently transfected with mEmerald-CD9 plasmid as described in Materials and Methods. Scale bar, 4 μm.

    Article Snippet: In this study, we used the following primary antibodies and dilutions: CD63 (1:3000; EXOAB-CD63A-1, System Biosciences), TSG101 (1:500; ab30871, Abcam), Alix (1:500; SC-53538, Santa Cruz Biotechnology), GRP94 (1:500; SC-393402, Santa Cruz Biotechnology), Lamin A (1:1000; ab26300, Abcam), and Histone H2B (1:500; ab1790, Abcam).

    Techniques: Staining, Mass Spectrometry, Expressing, Imaging, Stable Transfection, Transfection, Plasmid Preparation

    Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes. (A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N > C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C > N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    doi: 10.1371/journal.ppat.1005748

    Figure Lengend Snippet: Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes. (A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N > C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C > N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).

    Article Snippet: Antibodies The primary antibodies used in this work were as follows: mouse anti-HA (Covance, MMS-101P), mouse anti-IE1 ([ ], 1B12), mouse anti-IE1/IE2 (Millipore, MAB810R), mouse anti-Myc (Santa Cruz Biotechnology, sc-40), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, ab9485), rabbit anti-H2B (Abcam, ab1790), rabbit anti-STAT1 p84/p91 (Santa Cruz Biotechnology, sc-346), rabbit anti-STAT1α p91 (Santa Cruz Biotechnology, sc-345), rabbit anti-STAT2 (Santa Cruz Biotechnology, sc-22816), rabbit anti-STAT3 (Santa Cruz Biotechnology, sc-482x), rabbit anti-STAT3α (Cell Signaling Technologies, 8768), rabbit anti-pSTAT1 (Y701) (Cell Signaling Technologies, 9171), rabbit anti-pSTAT1 (S727) (Cell Signaling Technologies, 9177), rabbit anti-pSTAT3 (Cell Signaling Technologies, 9145), rabbit anti-SUMO1 (Santa Cruz Biotechnology, sc-9060) and mouse anti-α-tubulin (TUBA) (Thermo Fisher Scientific, A-11126).

    Techniques: Expressing, Immunofluorescence, Microscopy, Incubation, Staining, Chromatin Immunoprecipitation, Mutagenesis, Quantitative RT-PCR

    UBR7 is a histone H2B ubiquitin ligase. a Schematic of the domain organization of UBR7. b Immunoblots showing the in vitro interaction of purified UBR7 with recombinant histones H3, H4, H2B, and H2A. c Interaction of UBR7 full-length wild-type (WT) or catalytic-mutant (CM) with recombinant H2B, H2A/H2B dimer, core octamer, or purified nucleosomes from HeLa cells. d Ex vivo interaction of H2B with UBR7 WT or CM in HEK293T cells. e Ex vivo interaction of UBR7 with H2B WT or H2B mutant (K120R) in HEK293T cells. f , g In vitro ubiquitination assay with recombinant H2B ( f ), or purified nucleosomes from HeLa cells ( g )

    Journal: Nature Communications

    Article Title: Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor

    doi: 10.1038/s41467-019-08986-5

    Figure Lengend Snippet: UBR7 is a histone H2B ubiquitin ligase. a Schematic of the domain organization of UBR7. b Immunoblots showing the in vitro interaction of purified UBR7 with recombinant histones H3, H4, H2B, and H2A. c Interaction of UBR7 full-length wild-type (WT) or catalytic-mutant (CM) with recombinant H2B, H2A/H2B dimer, core octamer, or purified nucleosomes from HeLa cells. d Ex vivo interaction of H2B with UBR7 WT or CM in HEK293T cells. e Ex vivo interaction of UBR7 with H2B WT or H2B mutant (K120R) in HEK293T cells. f , g In vitro ubiquitination assay with recombinant H2B ( f ), or purified nucleosomes from HeLa cells ( g )

    Article Snippet: Cells were cross-linked with 1% formaldehyde and the chromatin was sheared and immunoprecipitated with the UBR7 antibody (Bethyl Laboratories), H2BK120Ub antibody (Millipore), H2B antibody (Abcam), or as a negative control IgG.

    Techniques: Western Blot, In Vitro, Purification, Recombinant, Mutagenesis, Ex Vivo, Ubiquitin Assay

    UBR7 is downregulated in invasive breast cancer cells. a Immunoblots of MCF10A, MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells to monitor expression of UBR7, H2BK120Ub, and H2B. ACTIN was used as a loading control. b Immunoblots for UBR7, H2BK120Ub, H2B, and ACTIN (loading control) in MCF10A cells expressing scrambled (SCR), UBR7-sh1, or UBR7-sh2 shRNA. c – e Immunoblots for UBR7, H2BK120Ub, H2B, and ACTIN (loading control) in MCF10A UBR7-sh1 ( c ), MDA-MB-231 ( d ), and MDA-MB-468 ( e ) cells expressing wild-type (UBR7-WT) and catalytic-mutant (UBR7-CM) UBR7. ACTIN was used as a loading control. f Venn diagram showing overlap of total H2BK120Ub binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. g Bar plot for quantitative PCR (qPCR) enrichment of UBR7 chromatin immunoprecipitation (ChIP) in MCF10A cells for selected genes. GAPDH was used as a negative control. h Average genebody density plot for H2BK120Ub binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. i Bar plot for qPCR enrichment of H2BK120Ub ChIP in MCF10A cells expressing SCR or UBR7-sh1. GAPDH was used as a negative control. j Average genebody density plot for H3K79me2 binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. k Emission parameter for a 10-state chromatin state model called by the default parameters of ChromHMM. States in the left column were annotated based on their closeness to the nearest transcription start sites (TSS) and nature of constituent marks. l Overlap enrichment analysis displaying chromatin state transitions between MCF10A control (SCR) cells ( Y -axis) and MCF10A UBR7-sh1 cells ( X -axis). The most significant state transitions include losses of H2BK120Ub/H3K79me2 low (States 1 to 5), H2BK120Ub/H3K79me2 high (States 2 to 1 or 3) and H3K79me2 only (States 3 to 5), which are highlighted by red circles. In g , i , error bars indicate standard deviation (s.d.); n = 3 technical replicates of a representative experiment (out of three experiments). P values were calculated using two-tailed t tests. * P

    Journal: Nature Communications

    Article Title: Atypical plant homeodomain of UBR7 functions as an H2BK120Ub ligase and breast tumor suppressor

    doi: 10.1038/s41467-019-08986-5

    Figure Lengend Snippet: UBR7 is downregulated in invasive breast cancer cells. a Immunoblots of MCF10A, MCF7, T47D, MDA-MB-231, and MDA-MB-468 cells to monitor expression of UBR7, H2BK120Ub, and H2B. ACTIN was used as a loading control. b Immunoblots for UBR7, H2BK120Ub, H2B, and ACTIN (loading control) in MCF10A cells expressing scrambled (SCR), UBR7-sh1, or UBR7-sh2 shRNA. c – e Immunoblots for UBR7, H2BK120Ub, H2B, and ACTIN (loading control) in MCF10A UBR7-sh1 ( c ), MDA-MB-231 ( d ), and MDA-MB-468 ( e ) cells expressing wild-type (UBR7-WT) and catalytic-mutant (UBR7-CM) UBR7. ACTIN was used as a loading control. f Venn diagram showing overlap of total H2BK120Ub binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. g Bar plot for quantitative PCR (qPCR) enrichment of UBR7 chromatin immunoprecipitation (ChIP) in MCF10A cells for selected genes. GAPDH was used as a negative control. h Average genebody density plot for H2BK120Ub binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. i Bar plot for qPCR enrichment of H2BK120Ub ChIP in MCF10A cells expressing SCR or UBR7-sh1. GAPDH was used as a negative control. j Average genebody density plot for H3K79me2 binding sites in Control (SCR) and UBR7-sh1-expressing MCF10A cells. k Emission parameter for a 10-state chromatin state model called by the default parameters of ChromHMM. States in the left column were annotated based on their closeness to the nearest transcription start sites (TSS) and nature of constituent marks. l Overlap enrichment analysis displaying chromatin state transitions between MCF10A control (SCR) cells ( Y -axis) and MCF10A UBR7-sh1 cells ( X -axis). The most significant state transitions include losses of H2BK120Ub/H3K79me2 low (States 1 to 5), H2BK120Ub/H3K79me2 high (States 2 to 1 or 3) and H3K79me2 only (States 3 to 5), which are highlighted by red circles. In g , i , error bars indicate standard deviation (s.d.); n = 3 technical replicates of a representative experiment (out of three experiments). P values were calculated using two-tailed t tests. * P

    Article Snippet: Cells were cross-linked with 1% formaldehyde and the chromatin was sheared and immunoprecipitated with the UBR7 antibody (Bethyl Laboratories), H2BK120Ub antibody (Millipore), H2B antibody (Abcam), or as a negative control IgG.

    Techniques: Western Blot, Multiple Displacement Amplification, Expressing, shRNA, Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Standard Deviation, Two Tailed Test

    SMC 5 functions jointly with DDX 11 to promote DNA repair Left panel: Growth curve was carried out to test ddx11 smc5‐aid cell proliferation after SMC5‐AID depletion induced by auxin treatment. Data represent means ± SD of three experiments. Experiments were carried out at 39.5°C. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Tubulin was used as loading control. Survival curve of ddx11 smc5‐aid cells treated with different concentrations of cisplatin after SMC5 depletion upon auxin treatment. Data represent means ± SD of four experiments. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Histone H2B was used as loading control.

    Journal: EMBO Reports

    Article Title: SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability

    doi: 10.15252/embr.201948222

    Figure Lengend Snippet: SMC 5 functions jointly with DDX 11 to promote DNA repair Left panel: Growth curve was carried out to test ddx11 smc5‐aid cell proliferation after SMC5‐AID depletion induced by auxin treatment. Data represent means ± SD of three experiments. Experiments were carried out at 39.5°C. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Tubulin was used as loading control. Survival curve of ddx11 smc5‐aid cells treated with different concentrations of cisplatin after SMC5 depletion upon auxin treatment. Data represent means ± SD of four experiments. Right panel: Western blot monitoring SMC5‐AID‐FLAG protein level in the presence or absence of auxin. Histone H2B was used as loading control.

    Article Snippet: Antibodies As primary antibodies for DT40 cell extracts, we used the following: anti‐α‐tubulin, mouse monoclonal antibody, clone B‐5‐1‐2 (Cat. No. T5168, Sigma‐Aldrich), 1:5,000 for WB; anti‐CHK1 (G‐4), mouse monoclonal antibody (Cat. No. sc‐8408, Santa Cruz Biotechnology), 1:1,000 for WB; anti‐CHK1‐P S345, rabbit polyclonal antibody (Cat. No. 133D3, #2341, Cell Signaling), 1:1,000 for WB; anti‐FANCD2, rabbit polyclonal antibody, the Takata laboratory, Kyoto medical University , 1:4,000 in BSA 5% TBS‐T 0.1% for WB; anti‐Flag M2, mouse monoclonal Ab (Cat. No. F1365 Sigma‐Aldrich), 1:3,000 for WB; anti‐Histone H2B, rabbit polyclonal (Cat. No. ab1790, Abcam), 1:3,000 for WB; anti‐HA, rat monoclonal (3F10, Cat. No. 11867423001, Roche), 1:500 for WB; and anti‐BrdU antibody, mouse monoclonal (B44, Cat. No. 5295722, BW), 1:5 for FACS.

    Techniques: Western Blot

    Post-translational modification of histones in NETs. (A) Summary of the post-translational modifications to the N-terminal tails of histones H1, H2A, H2B, H3, and H4 in RA and SLE neutrophil NETs (C, citrullination; A, acetylation; M, methylation; D, dimethylation). (B) Representative immunofluorescent staining of DNA (blue), citrullinated histone H3 (green) and PADI4 (red) in PMA- and A23187-induced NETs. Images taken on an Epifluorescent microscope (Zeiss).

    Journal: Frontiers in Immunology

    Article Title: Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2019.00423

    Figure Lengend Snippet: Post-translational modification of histones in NETs. (A) Summary of the post-translational modifications to the N-terminal tails of histones H1, H2A, H2B, H3, and H4 in RA and SLE neutrophil NETs (C, citrullination; A, acetylation; M, methylation; D, dimethylation). (B) Representative immunofluorescent staining of DNA (blue), citrullinated histone H3 (green) and PADI4 (red) in PMA- and A23187-induced NETs. Images taken on an Epifluorescent microscope (Zeiss).

    Article Snippet: Materials HetaSep solution and EasySep Human Neutrophil enrichment kit were from StemCell (Cambridge, UK); Ficoll-Paque was from GE Healthcare (Little Chalfont, UK); RPMI 1640 media plus without phenol red, L-glutamine, 25 mM HEPES and Hanks Balanced Salt Solution (HBSS), Annexin V-FITC, anti-rabbit AlexaFluor488, and anti-mouse AlexaFluor647 were from Life Technologies (Paisley, UK); Rapid Romanowsky stain was from TCS Biosciences (Botolph Claydon, UK); anti-CD16-FITC was from BD Biosciences (Oxford, UK); Propidium Iodide, A23187, phorbol 12-myristate 13-acetate (PMA), R848, LPS, DAPI, Mowiol 4–88, micrococcal nuclease, poly-L-lysine, human AB serum and PhastGel® Blue R Pre-measured tablets were from Sigma (Gillingham, UK); rabbit anti-neutrophil elastase antibody, mouse anti-myeloperoxidase antibody, rabbit anti-CRISP3 antibody, rabbit anti-thymidine phosphorylase antibody, rabbit anti-histone H2B, and mouse anti-PADI4 antibody were from Abcam (Cambridge, UK); goat anti-MMP8 antibody, mouse anti-CAMP (LL37) antibody, mouse anti-LCN1 antibody, and mouse anti-histone H1.0 antibody were from Biotechne (Abingdon, UK); Quantifluor dsDNA kit was from Promega (Southampton, UK); 96-well black plates, lithium-heparin vacutainers and Z-serum clot activator vacutainers were from Greiner (Stonehouse, UK); SYTOX® Green Nucleic Acid Stain and 0.5 M UltraPure EDTA pH8.0 was from ThermoFisher (Loughborough, UK); Strataclean beads were from Agilent (Cheadle, UK), cover slips were from Fischer Scientific (Loughborough, UK); MALP-2 was from Enzo Life Sciences (Exeter, UK); TNFα was from Merck (Watford, UK).

    Techniques: Modification, Methylation, Staining, Microscopy

    Immunofluorescent staining of NET proteins in RA and SLE NETs produced in response to A23187 or PMA. (A) Proteomics data is shown as box-and-whisker plots alongside (B) representative immunofluorescent images. Box-and-whisker plots also indicate the median (horizontal line) with dots representing each individual NET proteomics sample. DNA is stained with DAPI (blue), proteins are stained with antibodies as indicated on the figure (LL37/Cathelicidin, LCN2/Lipocalin, MMP8, Histone H1.0, PAD4 stained with AlexaFluor 647 and shown in red; CRISP3, Thymidine phosphorylase, Histone H2B stained with AlexaFluor 488 and shown in green). Images taken on a confocal laser-scanning microscope (Leica DM2500) at X20.

    Journal: Frontiers in Immunology

    Article Title: Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus

    doi: 10.3389/fimmu.2019.00423

    Figure Lengend Snippet: Immunofluorescent staining of NET proteins in RA and SLE NETs produced in response to A23187 or PMA. (A) Proteomics data is shown as box-and-whisker plots alongside (B) representative immunofluorescent images. Box-and-whisker plots also indicate the median (horizontal line) with dots representing each individual NET proteomics sample. DNA is stained with DAPI (blue), proteins are stained with antibodies as indicated on the figure (LL37/Cathelicidin, LCN2/Lipocalin, MMP8, Histone H1.0, PAD4 stained with AlexaFluor 647 and shown in red; CRISP3, Thymidine phosphorylase, Histone H2B stained with AlexaFluor 488 and shown in green). Images taken on a confocal laser-scanning microscope (Leica DM2500) at X20.

    Article Snippet: Materials HetaSep solution and EasySep Human Neutrophil enrichment kit were from StemCell (Cambridge, UK); Ficoll-Paque was from GE Healthcare (Little Chalfont, UK); RPMI 1640 media plus without phenol red, L-glutamine, 25 mM HEPES and Hanks Balanced Salt Solution (HBSS), Annexin V-FITC, anti-rabbit AlexaFluor488, and anti-mouse AlexaFluor647 were from Life Technologies (Paisley, UK); Rapid Romanowsky stain was from TCS Biosciences (Botolph Claydon, UK); anti-CD16-FITC was from BD Biosciences (Oxford, UK); Propidium Iodide, A23187, phorbol 12-myristate 13-acetate (PMA), R848, LPS, DAPI, Mowiol 4–88, micrococcal nuclease, poly-L-lysine, human AB serum and PhastGel® Blue R Pre-measured tablets were from Sigma (Gillingham, UK); rabbit anti-neutrophil elastase antibody, mouse anti-myeloperoxidase antibody, rabbit anti-CRISP3 antibody, rabbit anti-thymidine phosphorylase antibody, rabbit anti-histone H2B, and mouse anti-PADI4 antibody were from Abcam (Cambridge, UK); goat anti-MMP8 antibody, mouse anti-CAMP (LL37) antibody, mouse anti-LCN1 antibody, and mouse anti-histone H1.0 antibody were from Biotechne (Abingdon, UK); Quantifluor dsDNA kit was from Promega (Southampton, UK); 96-well black plates, lithium-heparin vacutainers and Z-serum clot activator vacutainers were from Greiner (Stonehouse, UK); SYTOX® Green Nucleic Acid Stain and 0.5 M UltraPure EDTA pH8.0 was from ThermoFisher (Loughborough, UK); Strataclean beads were from Agilent (Cheadle, UK), cover slips were from Fischer Scientific (Loughborough, UK); MALP-2 was from Enzo Life Sciences (Exeter, UK); TNFα was from Merck (Watford, UK).

    Techniques: Staining, Produced, Whisker Assay, Laser-Scanning Microscopy

    Subcellular localization of stably expressed Ptc1. (A) Schematic representation of stably transfected full-length human Ptc1 protein with possess 12 transmembrane domains. Red box at N-terminus indicates 3×T7-tag, green box at end of intracellular domain indicates C-terminal 3×FLAG-tag, respectively. (B) Western blot analyses of Ptc1 protein stably expressed in HeLa cells. Ptc1 immunoprecipitated from cell extracts with C-terminal FLAG-tag and immunoblotted with anti-N-terminal T7 tag antibody (left panel) or anti-C-terminal FLAG tag antibody (right panel). Anti-T7 blot detects doublet bands corresponding to full-length Ptc1 (Ptc1 FL), while anti-FLAG blot reveals small C-terminal fragments representing the intracellular domain of Ptc1 (ICD7 fragments) as well as Ptc1 FL. (C) Multiple staining of Ptc1-stably expressing cells by anti-T7 tag immunostain (a: Ptc1 N-terminus), anti-FLAG-tag immunostain (b: Ptc1 C-terminus), DAPI DNA stain (c: nucleus) and their merged image (d). Scale bar, 20 µm. (D) Cell fractionation analysis of Ptc1-stably expressing cells. BIP and Histone H2B used as cytoplasmic and nuclear marker, respectively. Ptc1 FL (+), cells stably expressing full-length Ptc1; Ptc1 FL (−), control HeLa cells.

    Journal: PLoS ONE

    Article Title: A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

    doi: 10.1371/journal.pone.0018638

    Figure Lengend Snippet: Subcellular localization of stably expressed Ptc1. (A) Schematic representation of stably transfected full-length human Ptc1 protein with possess 12 transmembrane domains. Red box at N-terminus indicates 3×T7-tag, green box at end of intracellular domain indicates C-terminal 3×FLAG-tag, respectively. (B) Western blot analyses of Ptc1 protein stably expressed in HeLa cells. Ptc1 immunoprecipitated from cell extracts with C-terminal FLAG-tag and immunoblotted with anti-N-terminal T7 tag antibody (left panel) or anti-C-terminal FLAG tag antibody (right panel). Anti-T7 blot detects doublet bands corresponding to full-length Ptc1 (Ptc1 FL), while anti-FLAG blot reveals small C-terminal fragments representing the intracellular domain of Ptc1 (ICD7 fragments) as well as Ptc1 FL. (C) Multiple staining of Ptc1-stably expressing cells by anti-T7 tag immunostain (a: Ptc1 N-terminus), anti-FLAG-tag immunostain (b: Ptc1 C-terminus), DAPI DNA stain (c: nucleus) and their merged image (d). Scale bar, 20 µm. (D) Cell fractionation analysis of Ptc1-stably expressing cells. BIP and Histone H2B used as cytoplasmic and nuclear marker, respectively. Ptc1 FL (+), cells stably expressing full-length Ptc1; Ptc1 FL (−), control HeLa cells.

    Article Snippet: The following antibodies were used for immunological analyses in this study: anti-FLAG polyclonal (Sigma), anti-FLAG M2 monoclonal (Sigma), anti-T7 tagR monoclonal (Novagen), anti-Ptc-ICD7 C-terminal (1420–1434) (prepared in this study), anti-human Ptc1 SSD (500–600) (Abcam, Cat. No. ab39266), anti-human Ptc1 ICD7 peptide (1271- HPESRHHPPSNPRQQ-1285 ) antibody (Abcam, Cat. No. 51983), anti-TRA-2 ICD7 , anti-tubulin (ICN), anti-BIP (BD Bioscience), anti-Histone H2B (Santa Cruz), Alexa FluorR 488 anti-rabbit IgG (Invitrogen), Alexa FluorR 488 anti-mouse IgG (Invitrogen), Alexa FluorR 568 anti-mouse IgG (Invitrogen), HRP-conjugated anti-rabbit IgG (GE healthcare), and HRP-conjugated anti-mouse IgG (GE healthcare).

    Techniques: Stable Transfection, Transfection, Western Blot, Immunoprecipitation, FLAG-tag, Staining, Expressing, Cell Fractionation, Marker

    Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with H2B wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: Depletion of CRL7 SMU1 complex proteins induce mitotic defects. (A) HeLa cells were transfected with either control or SMU1 siRNAs. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 10 μm. (B) Quantification of results shown in A ( n =50 cells each). (C) HeLa cells were transduced with control shRNA, CUL7 shRNA, DDB1 shRNA or RNF40 shRNA. Cells were stained with antibody against α-tubulin to check the spindle defects (multipolar spindles) and nuclei were counterstained with DAPI to check for chromosomal defects (lagging chromosomes and anaphase bridges). Scale bar: 5 μm. (D) Quantification of results shown in C ( n =40 cells each). (E) Cells were transfected with H2B wild type (WT) and H2B K120R mutant. (F) Various mitotic abnormalities in cells expressing H2B WT and H2B K120R mutant were checked using immunofluorescence after staining with antibody against α-tubulin and DAPI. Scale bar: 5 μm. (G) Quantification of results shown in F ( n =50 cells each). Error bars indicate the mean+s.d.; *** P

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Transfection, Staining, Transduction, shRNA, Mutagenesis, Expressing, Immunofluorescence

    CRL7 SMU1 complex is necessary for driving SMC1 gene expression. (A) Exponentially growing HeLa cells were subjected to ChIP analysis using either anti-SMU1 or anti-IgG antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three independent experiments. (B) Cells expressing control shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Fold change of H2Bub enrichment at indicated loci with respect to control shRNA is shown. The data shown is derived from three independent experiments. (C) Total RNA was extracted from HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and expression levels of various genes measured by qRT-PCR from three independent experiments is shown. (D) HeLa cells transduced with control, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by immunoblotting with specific antibody. (E) HeLa cells were transfected with H2B wild type (WT) and H2B K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three independent experiments was plotted. Error bars indicate the mean+s.d; *** P

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: CRL7 SMU1 complex is necessary for driving SMC1 gene expression. (A) Exponentially growing HeLa cells were subjected to ChIP analysis using either anti-SMU1 or anti-IgG antibody. SMU1 enrichment at various loci is shown. The data shown is derived from three independent experiments. (B) Cells expressing control shRNA, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA and SMU1 shRNA were subjected to ChIP analysis using H2Bub antibody. Fold change of H2Bub enrichment at indicated loci with respect to control shRNA is shown. The data shown is derived from three independent experiments. (C) Total RNA was extracted from HeLa cells transfected with control or SMU1 siRNA or CUL7 shRNA or DDB1 shRNA and RNF40 shRNA, and expression levels of various genes measured by qRT-PCR from three independent experiments is shown. (D) HeLa cells transduced with control, CUL7 shRNA, DDB1 shRNA, RNF40 shRNA or SMU1 shRNA and levels of SMC1a protein was measured by immunoblotting with specific antibody. (E) HeLa cells were transfected with H2B wild type (WT) and H2B K120R mutant. Relative expression of indicated genes measured by using qRT-PCR from three independent experiments was plotted. Error bars indicate the mean+s.d; *** P

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Expressing, Chromatin Immunoprecipitation, Derivative Assay, shRNA, Transfection, Quantitative RT-PCR, Transduction, Mutagenesis

    CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Journal: Journal of Cell Science

    Article Title: CRL7SMU1 E3 ligase complex-driven H2B ubiquitylation functions in sister chromatid cohesion by regulating SMC1 expression

    doi: 10.1242/jcs.213868

    Figure Lengend Snippet: CRL7 SMU1 complex regulates the monoubiquitylation of H2B at position K120. (A) SFB-tagged CUL7, DDB1, RNF40, SMU1, Rab7 or empty vector (EV) were transfected and interaction of H2B was detected by immunoblotting with specific antibody after streptavidin Sepharose pull-down. (B) HeLa cells were transduced with either control or SMU1-specific shRNA followed by overexpression of SFB-tagged RNF40. 72 h post transduction, pull-down was performed with streptavidin Sepharose beads, and interaction of DDB1 and H2B with RNF40 was evaluated by immunoblotting with their respective antibodies. (C) SFB-tagged SMU1 was overexpressed in cells transduced with either control or RNF40-specific shRNA. The interaction of SMU1 with H2B and DDB1 was detected through immunoblotting using specific antibodies after immunoprecipitation. (D,E) Cells were transduced with either control or DDB1 shRNA (E), and control or CUL7 shRNA containing viral particles. Pull-down followed by detection of different indicated proteins in precipitates were done as described in B. (F) Model shows the assembly of CRL7 SMU1 complex in association with its substrate H2B. (G) GST pull-down assay was performed with immobilized control GST or GST–SMU1 fusion proteins on glutathione beads, followed by incubation with bacterially purified MBP-H2B. The interaction of SMU1 with H2B was assessed by immunoblotting with anti-MBP antibody. Expression of GST, recombinant GST-SMU1 and MBP-H2B was shown by Coomassie Blue staining. (H) HeLa cells were transduced using either control or RNF40 shRNA. Post 72 h, cells were collected and lysed to isolate soluble and histone fractions. Lysates were subjected to SDS-PAGE followed by immunoblotting using the indicated antibodies. (I) Cells were transfected/transduced with either control or SMU1 siRNA, (J) or DDB1 shRNA, (K) or CUL7 shRNA. Soluble and acid-extracted histone fractions were subjected to SDS-PAGE followed by immunoblotting using indicated antibodies. The data presented here represent three independent experiments.

    Article Snippet: Antibodies against SMU1 (Abgent #AT3965a; 1:1000); RNF40 (Sigma #R9029; 1:2000); CUL7 (Sigma #C1743; 1:2000); DDB1 (Bethyl #A300-462A; 1:5000); SMC1a (Abcam #ab133643; 1:1000); H2B (Millipore #07-371; 1:5000); histone H2B ubiquitylated at Lys120 (H2Bub; Cell Signaling Technology #5546; 1:1000); cyclin A (BD #611269; 1:1000); CDT1 (Bethyl #A300-786A; 1:1000); histone H3 phosphorylated at Ser10 (pH3; Cell Signaling Technology #9701L; for western blotting 1:1000, for immunofluorescence 1:200); Cul4a (Bethyl #A300-739A; 1:5000); RNF20 (Abcam #ab32629; 1:1000); Myc-tag (Santa Cruz #9E10; 1:1000); FLAG-tag (Sigma, #F3165; 1:10,000); HA (Bethyl, #A190-108A; 1:1000), actin (Sigma, #A5441; 1:10,000) and α-tubulin (Sigma, #T6074; for western blotting 1:5000, for immunofluorescence 1:200) were used in this study.

    Techniques: Plasmid Preparation, Transfection, Transduction, shRNA, Over Expression, Immunoprecipitation, Pull Down Assay, Incubation, Purification, Expressing, Recombinant, Staining, SDS Page

    Interaction between ASF1 and MCM2 is increased following DNA damage. A , U2OS cells expressing GFP-MCM2 were either mock treated (lane 1–2) or treated with etoposide at 50 μ m for 1 h and allowed to recover for either 1 h (lane 3–4) or 4 h (lane 5–6). Total cell extracts were immunoprecipitated with GFP-antibodies and immunoblotted with GFP (top) or ASF1A (bottom) antibodies. A similar experiment was performed, but instead comparing protein lysates prepared using either 1% triton X-100 buffer ( B ) or RIPA buffer ( C ). Immunoprecipitates (IP) were immunoblotted with antibodies recognizing GFP or ASF1A, and total cell lysates (TCL) were immunoblotted with antibodies recognizing histone H2AX phosphorylated on serine 139 and histone H2B.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage *

    doi: 10.1074/mcp.M115.048991

    Figure Lengend Snippet: Interaction between ASF1 and MCM2 is increased following DNA damage. A , U2OS cells expressing GFP-MCM2 were either mock treated (lane 1–2) or treated with etoposide at 50 μ m for 1 h and allowed to recover for either 1 h (lane 3–4) or 4 h (lane 5–6). Total cell extracts were immunoprecipitated with GFP-antibodies and immunoblotted with GFP (top) or ASF1A (bottom) antibodies. A similar experiment was performed, but instead comparing protein lysates prepared using either 1% triton X-100 buffer ( B ) or RIPA buffer ( C ). Immunoprecipitates (IP) were immunoblotted with antibodies recognizing GFP or ASF1A, and total cell lysates (TCL) were immunoblotted with antibodies recognizing histone H2AX phosphorylated on serine 139 and histone H2B.

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (Roche 11814460001), anti-MCM2 (Rabbit polyclonal, Abcam #Ab31159, Cambridge, MA), anti-MCM5 (Rabbit monoclonal, Abcam #Ab75975), anti-ASF1 (Rabbit monoclonal, Cell Signaling #C6E10, Danvers, MA), anti-γH2AX (Rabbit polyclonal, Santa Cruz #sc-101696, Dallas) and anti-H2B (Rabbit polyclonal, Cell Signaling #2722).

    Techniques: Expressing, Immunoprecipitation

    Global levels of O -GlcNAc glycosylation increase after T cell activation. ( A ) Primary human T cells were incubated with control beads or anti-CD3/CD28 beads and then analyzed for O -GlcNAc by flow cytometry. The staining intensity of stimulated cells relative to control cells at each time point is plotted (five donors over three independent experiments) along with the mean and 95% confidence interval of a linear regression analysis. ( B ) Representative line graphs are shown for one donor. ( C ) Whole-cell lysates from control and stimulated T cells were analyzed for O -GlcNAc levels by immunoblot with anti- O -GlcNAc Ab RL-2 or CTD110.6. GAPDH served as a loading control. A total of at least two donors over at least two independent experiments were analyzed with similar results for each blot. ( D ) Cytosolic (C) and nuclear (N) extracts from T cells incubated with control beads or anti-CD3/CD28 beads for 18 h were analyzed for O -GlcNAc levels by immunoblot with anti– O -GlcNAc Ab RL-2 or CTD110.6. GAPDH and histone H2B served as controls for loading and compartmentalization. A total of three donors over two independent experiments were analyzed with similar results for each blot.

    Journal: The Journal of Immunology Author Choice

    Article Title: Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

    doi: 10.4049/jimmunol.1502031

    Figure Lengend Snippet: Global levels of O -GlcNAc glycosylation increase after T cell activation. ( A ) Primary human T cells were incubated with control beads or anti-CD3/CD28 beads and then analyzed for O -GlcNAc by flow cytometry. The staining intensity of stimulated cells relative to control cells at each time point is plotted (five donors over three independent experiments) along with the mean and 95% confidence interval of a linear regression analysis. ( B ) Representative line graphs are shown for one donor. ( C ) Whole-cell lysates from control and stimulated T cells were analyzed for O -GlcNAc levels by immunoblot with anti- O -GlcNAc Ab RL-2 or CTD110.6. GAPDH served as a loading control. A total of at least two donors over at least two independent experiments were analyzed with similar results for each blot. ( D ) Cytosolic (C) and nuclear (N) extracts from T cells incubated with control beads or anti-CD3/CD28 beads for 18 h were analyzed for O -GlcNAc levels by immunoblot with anti– O -GlcNAc Ab RL-2 or CTD110.6. GAPDH and histone H2B served as controls for loading and compartmentalization. A total of three donors over two independent experiments were analyzed with similar results for each blot.

    Article Snippet: Anti-GAPDH (5174), anti-histone H2B (2934), anti-LCP1 (5350), anti-VIM (5741), anti–aryl hydrocarbon receptor nuclear translocator (ARNT; 5537), anti-HCLS1 (4503), anti-WNK1 (4979), anti-EWSR1 (11910), anti-ARID1A (12354), anti-MYPT1 (2634), anti-RUNX1 (4334), anti-SP1 (9389), and anti-NUP98 (2292) were purchased from Cell Signaling Technology.

    Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry, Staining

    Impairment of Aurora-B-mediated activities on H2B and HIPK2 induces cytokinesis failure. a , b HeLa cells were transfected with vectors carrying the indicated GFP-HIPK2 forms and stained 24 h post-transfection with anti-p-Histone H2B-S14 and Hoechst. The percentages of midbodies positive for H2B-S14 P staining in the transfected populations a and the percentages of binucleated cells also in the transfected populations b are reported as mean ± SD of two different experiments. c HF were transfected with vectors carrying GFP-tagged H2B-WT or the non-phosphorylatable H2B-S32A derivative. Cells were stained with anti-β-tubulin Ab and Hoechst. The percentages of binucleated cells in the GFP-positive populations were evaluated at the indicated time-points post-transfection and reported as fold change relative to that of GFP-H2B-WT in two different experiments. * p

    Journal: Oncogene

    Article Title: HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

    doi: 10.1038/s41388-018-0191-6

    Figure Lengend Snippet: Impairment of Aurora-B-mediated activities on H2B and HIPK2 induces cytokinesis failure. a , b HeLa cells were transfected with vectors carrying the indicated GFP-HIPK2 forms and stained 24 h post-transfection with anti-p-Histone H2B-S14 and Hoechst. The percentages of midbodies positive for H2B-S14 P staining in the transfected populations a and the percentages of binucleated cells also in the transfected populations b are reported as mean ± SD of two different experiments. c HF were transfected with vectors carrying GFP-tagged H2B-WT or the non-phosphorylatable H2B-S32A derivative. Cells were stained with anti-β-tubulin Ab and Hoechst. The percentages of binucleated cells in the GFP-positive populations were evaluated at the indicated time-points post-transfection and reported as fold change relative to that of GFP-H2B-WT in two different experiments. * p

    Article Snippet: The following Abs were employed: anti-Aurora-B (BD-Bioscience); anti-ECT2, anti-PRC1, anti-MKLP1, and anti-PLK1 (Santa Cruz); anti-HIPK2 (rabbit polyclonal Ab [ ]); anti-p-histone-H2B-S14 (Cell Signaling); anti-p-histone-H2B-S32, anti-H2B, anti-53BP1, and anti-MgcRacGAP1 (Abcam); anti-β-tubulin-Cy3 and anti-α-tubulin-FITC (Sigma-Aldrich); secondary 488- or 594-conjugated Abs (Life Technologies).

    Techniques: Transfection, Staining

    Aurora-B phosphorylates H2B at Ser32. a Cold in vitro kinase assays were performed and H2B-S32 P detected using anti-p-Histone H2B-Ser32 Ab. Ponceau staining and immunoblot with anti-H2B Ab were used as loading controls. n = three independent experiment. b Unsynchronized HeLa cells were treated with Hesperadin or DMSO for 80 min and stained with anti-p-Histone H2B-S32 and anti-β-tubulin Abs. Hoechst was used to stain DNA (blue). Representative images of cells at telophase stage are reported. Scale bar is 5 μm. c Representative confocal images of HeLa cells at indicated stages of mitosis and cytokinesis showing colocalization between H2B-S32 P (green) and Aurora-B (red) n = four independent experiment. DNA was stained with Red-Dot2 far-red (pseudo-colored blue); scale bar is 10 μm. d , e HeLa cells stably expressing GFP-H2B WT or its derivative GFP-H2B-S32A at passage two after the establishment of stable populations (i.e., 15 days after blastacidin treatment) were enriched in telophase and protein lysates were obtained from TCEs (T-TCE) or after midbody isolation (MI). Untrasfected HeLa cells were used as negative control. The indicated proteins were analyzed by WB. Immunoblots with anti-PRC1 and anti-p-Histone H2B-S14 Abs were used as positive control to verify the quality of midbody isolation. Immunoblot with anti-PCNA Ab was used as control to evaluate nuclear contamination. Representative WBs are shown. In d , samples were loaded on the same gel and processed on the same filter. Blot was vertically cropped to eliminate non-related samples

    Journal: Oncogene

    Article Title: HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

    doi: 10.1038/s41388-018-0191-6

    Figure Lengend Snippet: Aurora-B phosphorylates H2B at Ser32. a Cold in vitro kinase assays were performed and H2B-S32 P detected using anti-p-Histone H2B-Ser32 Ab. Ponceau staining and immunoblot with anti-H2B Ab were used as loading controls. n = three independent experiment. b Unsynchronized HeLa cells were treated with Hesperadin or DMSO for 80 min and stained with anti-p-Histone H2B-S32 and anti-β-tubulin Abs. Hoechst was used to stain DNA (blue). Representative images of cells at telophase stage are reported. Scale bar is 5 μm. c Representative confocal images of HeLa cells at indicated stages of mitosis and cytokinesis showing colocalization between H2B-S32 P (green) and Aurora-B (red) n = four independent experiment. DNA was stained with Red-Dot2 far-red (pseudo-colored blue); scale bar is 10 μm. d , e HeLa cells stably expressing GFP-H2B WT or its derivative GFP-H2B-S32A at passage two after the establishment of stable populations (i.e., 15 days after blastacidin treatment) were enriched in telophase and protein lysates were obtained from TCEs (T-TCE) or after midbody isolation (MI). Untrasfected HeLa cells were used as negative control. The indicated proteins were analyzed by WB. Immunoblots with anti-PRC1 and anti-p-Histone H2B-S14 Abs were used as positive control to verify the quality of midbody isolation. Immunoblot with anti-PCNA Ab was used as control to evaluate nuclear contamination. Representative WBs are shown. In d , samples were loaded on the same gel and processed on the same filter. Blot was vertically cropped to eliminate non-related samples

    Article Snippet: The following Abs were employed: anti-Aurora-B (BD-Bioscience); anti-ECT2, anti-PRC1, anti-MKLP1, and anti-PLK1 (Santa Cruz); anti-HIPK2 (rabbit polyclonal Ab [ ]); anti-p-histone-H2B-S14 (Cell Signaling); anti-p-histone-H2B-S32, anti-H2B, anti-53BP1, and anti-MgcRacGAP1 (Abcam); anti-β-tubulin-Cy3 and anti-α-tubulin-FITC (Sigma-Aldrich); secondary 488- or 594-conjugated Abs (Life Technologies).

    Techniques: In Vitro, Staining, Stable Transfection, Expressing, Isolation, Negative Control, Western Blot, Positive Control

    P2X6 subunit interacts with SF3A1 and Spectrin α2 in nuclear extracts from hippocampus of 8 month-old mice and alters splicing efficiency  in vitro . ( a ) Western blot of subcellular fractions from 8 month-old mice hippocampus. P2X6 subunit was located in both the cytosolic (cyt) and the nuclear fraction (nuc), as identified by localization with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NeuN (neuronal nuclei marker)/histone 2b (H2b), respectively. ( b ) Nuclear extracts immunoprecipitated with anti-P2X6 antibody were resolved in a 2D electrophoresis gel and selected spots were isolated, processed and identified by MALDI/TOF (n = 3 mice). ( c-f ) Nuclear extracts immunoprecipitated with either IgG or anti-P2X6 antibodies were analysed by immunoblotting with antibodies against SF3A1 ( c ) and spectrin α-2( d ). Nuclear extracts were also immunoprecipitated with spectrin α-2 ( e ) or SF3A1 ( f ) antibodies and immunoblotted with anti-P2X6 antibody. ( g ) Scheme shows four DNA constructions prepared for splicing activity quantitation and their respective protein translations. ( h ) Splicing inhibitor isoginkgetin (IGK) was used to validate the molecular tools employed to quantify the splicing efficiency. As expected TPI I firefly luciferase-dependent luminescence decreases and TPI II firefly luciferase-dependent luminescence increases when splicing is impaired (mean±s.e.m., n = 5 independent experiments, * P

    Journal: PLoS ONE

    Article Title: Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

    doi: 10.1371/journal.pone.0123121

    Figure Lengend Snippet: P2X6 subunit interacts with SF3A1 and Spectrin α2 in nuclear extracts from hippocampus of 8 month-old mice and alters splicing efficiency in vitro . ( a ) Western blot of subcellular fractions from 8 month-old mice hippocampus. P2X6 subunit was located in both the cytosolic (cyt) and the nuclear fraction (nuc), as identified by localization with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NeuN (neuronal nuclei marker)/histone 2b (H2b), respectively. ( b ) Nuclear extracts immunoprecipitated with anti-P2X6 antibody were resolved in a 2D electrophoresis gel and selected spots were isolated, processed and identified by MALDI/TOF (n = 3 mice). ( c-f ) Nuclear extracts immunoprecipitated with either IgG or anti-P2X6 antibodies were analysed by immunoblotting with antibodies against SF3A1 ( c ) and spectrin α-2( d ). Nuclear extracts were also immunoprecipitated with spectrin α-2 ( e ) or SF3A1 ( f ) antibodies and immunoblotted with anti-P2X6 antibody. ( g ) Scheme shows four DNA constructions prepared for splicing activity quantitation and their respective protein translations. ( h ) Splicing inhibitor isoginkgetin (IGK) was used to validate the molecular tools employed to quantify the splicing efficiency. As expected TPI I firefly luciferase-dependent luminescence decreases and TPI II firefly luciferase-dependent luminescence increases when splicing is impaired (mean±s.e.m., n = 5 independent experiments, * P

    Article Snippet: Antibodies used were: P2X6 (Chemicon, 1:200), P2X2 (1:200), P2X4 (1:1000), GFP (1:1000), c-myc (1:1000), spectrin α2 (1:200), histone 2b (Cell Signalling, 1:1000) and α-tubulin (Sigma Aldrich, 1:10000).

    Techniques: Mouse Assay, In Vitro, Western Blot, Marker, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Isolation, Activity Assay, Quantitation Assay, Luciferase

    Chl1 expression and chromatin binding are induced during S-phase. A) Logarithmically growing cells expressing Chl1-13Myc (YBS 1129) were harvested and analyzed for chromatin binding with or without MMS exposure. Immunoblots show whole cells extracts (WCE), cytoplasmic fractions (Cyt) and chromatin bound fractions (CB). Histone 2B (H2B) and Phosphoglycerate Kinase (PGK) were probed in parallel as positive controls for chromatin-bound and cytoplasmic proteins, respectively. B) Logarithmically growing cells expressing Chl1-13MYC were synchronized in G1 (alpha factor) and released into fresh medium. Samples were harvested every 15minutes and analyzed by Immunoblotting for Chl1-13MYC and Swi6 as a loading control. Parallel blots were also analyzed for Histone 2B (H2B) and Phosphoglyerinkinase (PGK) to confirm chromatin and cytoplasmic fractions, respectively. C) G1 synchronized cells expressing Chl1-13Myc were released in fresh medium and samples collected every 15 minutes, processed for chromatin binding and probed to detect Chl1-13MYC and Swi6 (loading control). D) Data shown are Chl1 protein levels relative to Swi6 (blue line) and chromatin enrichment relative to Swi6 (red line) over the cell cycle averaged from 3 independent experiments. Shaded portion denotes S-phase. E) Flow cytometric data for cells analyzed in B) and C).

    Journal: PLoS ONE

    Article Title: Chl1 DNA Helicase Regulates Scc2 Deposition Specifically during DNA-Replication in Saccharomyces cerevisiae

    doi: 10.1371/journal.pone.0075435

    Figure Lengend Snippet: Chl1 expression and chromatin binding are induced during S-phase. A) Logarithmically growing cells expressing Chl1-13Myc (YBS 1129) were harvested and analyzed for chromatin binding with or without MMS exposure. Immunoblots show whole cells extracts (WCE), cytoplasmic fractions (Cyt) and chromatin bound fractions (CB). Histone 2B (H2B) and Phosphoglycerate Kinase (PGK) were probed in parallel as positive controls for chromatin-bound and cytoplasmic proteins, respectively. B) Logarithmically growing cells expressing Chl1-13MYC were synchronized in G1 (alpha factor) and released into fresh medium. Samples were harvested every 15minutes and analyzed by Immunoblotting for Chl1-13MYC and Swi6 as a loading control. Parallel blots were also analyzed for Histone 2B (H2B) and Phosphoglyerinkinase (PGK) to confirm chromatin and cytoplasmic fractions, respectively. C) G1 synchronized cells expressing Chl1-13Myc were released in fresh medium and samples collected every 15 minutes, processed for chromatin binding and probed to detect Chl1-13MYC and Swi6 (loading control). D) Data shown are Chl1 protein levels relative to Swi6 (blue line) and chromatin enrichment relative to Swi6 (red line) over the cell cycle averaged from 3 independent experiments. Shaded portion denotes S-phase. E) Flow cytometric data for cells analyzed in B) and C).

    Article Snippet: Whole cell extract, cytoplasmic and chromatin bound fractions were resolved by SDS-PAGE electrophoresis and analyzed by Western blot using anti-MYC 9E10 (1:1000) (Santa Cruz), anti-HA (1:500) (f7) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) or by anti-Histone 2B (1:2000) (Santa Cruz) in combination with goat anti Rabbit HRP (1:10,000) or by anti-phosphoglycerate kinase (Invitrogen) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) and ECL plus (GE healthcare) for visualization.

    Techniques: Expressing, Binding Assay, Western Blot, Flow Cytometry

    Cells lacking Chl1, but not Fen1, exhibit reduced binding of cohesins to chromatin. A) Logarithmically growing wild type (YBS 1157) and chl1 (YBS 1175) expressing Mcd1-6HA processed for Mcd1 chromatin binding. Whole cell extracts (WCE), Cytoplasmic fractions (Cyt) and Chromatin bound fractions (CB) shown. Histone 2B (H2B) and Phosphoglycerate kinase (PGK) shown as controls for cytoplasmic and chromatin-bound proteins, respectively. B) Quantification of Mcd1 binding to chromatin in chl1 mutant cells compared to wildtype levels (normalized to 100%). Mcd1 enrichment to DNA based on the ratio of Mcd1-6HA to Histone 2B levels obtained from 3 independent experiments. C and D) Experimental analysis of fen1 mutant cells (YSR 117) identical to that shown in A and B for chl1 mutant cells. E) Enrichment of Mcd1-6HA in chl1 mutant cells at five independent chromosome arm CAR sites along Chromosome III compared to levels obtained from wild type cells (normalized to 100%). F) Enrichment of Mcd1-6HA as shown in E) except for five centromere ( CEN ) sites.

    Journal: PLoS ONE

    Article Title: Chl1 DNA Helicase Regulates Scc2 Deposition Specifically during DNA-Replication in Saccharomyces cerevisiae

    doi: 10.1371/journal.pone.0075435

    Figure Lengend Snippet: Cells lacking Chl1, but not Fen1, exhibit reduced binding of cohesins to chromatin. A) Logarithmically growing wild type (YBS 1157) and chl1 (YBS 1175) expressing Mcd1-6HA processed for Mcd1 chromatin binding. Whole cell extracts (WCE), Cytoplasmic fractions (Cyt) and Chromatin bound fractions (CB) shown. Histone 2B (H2B) and Phosphoglycerate kinase (PGK) shown as controls for cytoplasmic and chromatin-bound proteins, respectively. B) Quantification of Mcd1 binding to chromatin in chl1 mutant cells compared to wildtype levels (normalized to 100%). Mcd1 enrichment to DNA based on the ratio of Mcd1-6HA to Histone 2B levels obtained from 3 independent experiments. C and D) Experimental analysis of fen1 mutant cells (YSR 117) identical to that shown in A and B for chl1 mutant cells. E) Enrichment of Mcd1-6HA in chl1 mutant cells at five independent chromosome arm CAR sites along Chromosome III compared to levels obtained from wild type cells (normalized to 100%). F) Enrichment of Mcd1-6HA as shown in E) except for five centromere ( CEN ) sites.

    Article Snippet: Whole cell extract, cytoplasmic and chromatin bound fractions were resolved by SDS-PAGE electrophoresis and analyzed by Western blot using anti-MYC 9E10 (1:1000) (Santa Cruz), anti-HA (1:500) (f7) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) or by anti-Histone 2B (1:2000) (Santa Cruz) in combination with goat anti Rabbit HRP (1:10,000) or by anti-phosphoglycerate kinase (Invitrogen) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) and ECL plus (GE healthcare) for visualization.

    Techniques: Binding Assay, Expressing, Mutagenesis

    Chl1 regulates the binding of Scc2 to chromatin. A) Logarithmically growing wild type (YSR 135) and chl1 (YSR 138) expressing Scc2-3HA processed for Scc2 chromatin binding. Whole cell extracts (WCE), Cytoplasmic fractions (Cyt) and Chromatin bound fractions (CB) shown. Histone 2B (H2B) and Phosphoglycerate kinase (PGK) shown as controls for cytoplasmic and chromatin-bound proteins, respectively. B) Quantification of Scc2 binding to chromatin in chl1 mutant cells compared to wildtype levels (normalized to 100%). Scc2-3HA enrichment calculated from 3 independent experiments. C) Enrichment of Scc3-3HA in chl1 mutant cells at five independent chromosome arm CAR sites along Chromosome III compared to levels obtained from wild type cells (normalized to 100%). D) Enrichment of Scc2-3HA as shown in C) except for five sites that map across centromere III ( CEN ).

    Journal: PLoS ONE

    Article Title: Chl1 DNA Helicase Regulates Scc2 Deposition Specifically during DNA-Replication in Saccharomyces cerevisiae

    doi: 10.1371/journal.pone.0075435

    Figure Lengend Snippet: Chl1 regulates the binding of Scc2 to chromatin. A) Logarithmically growing wild type (YSR 135) and chl1 (YSR 138) expressing Scc2-3HA processed for Scc2 chromatin binding. Whole cell extracts (WCE), Cytoplasmic fractions (Cyt) and Chromatin bound fractions (CB) shown. Histone 2B (H2B) and Phosphoglycerate kinase (PGK) shown as controls for cytoplasmic and chromatin-bound proteins, respectively. B) Quantification of Scc2 binding to chromatin in chl1 mutant cells compared to wildtype levels (normalized to 100%). Scc2-3HA enrichment calculated from 3 independent experiments. C) Enrichment of Scc3-3HA in chl1 mutant cells at five independent chromosome arm CAR sites along Chromosome III compared to levels obtained from wild type cells (normalized to 100%). D) Enrichment of Scc2-3HA as shown in C) except for five sites that map across centromere III ( CEN ).

    Article Snippet: Whole cell extract, cytoplasmic and chromatin bound fractions were resolved by SDS-PAGE electrophoresis and analyzed by Western blot using anti-MYC 9E10 (1:1000) (Santa Cruz), anti-HA (1:500) (f7) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) or by anti-Histone 2B (1:2000) (Santa Cruz) in combination with goat anti Rabbit HRP (1:10,000) or by anti-phosphoglycerate kinase (Invitrogen) in combination with goat anti mouse HRP (1:10,000) (Bio-Rad) and ECL plus (GE healthcare) for visualization.

    Techniques: Binding Assay, Expressing, Mutagenesis

    WEE1 pY37-H2B epigenetic signaling axis downregtulates IDH2 mRNA expression and 5-hmC levels in cancer cell; a model WEE1 overexpressing cancer cells deposits pY37-H2B repressive marks to suppress the expression of IDH2 transcription which is reflected in a significant loss of global 5-hmC levels that promotes gene expression programs that favor tumor growth. Inhibition of WEE1 by AZD1775 led to the loss of pY37-H2B which in turn restored 5-hmC levels, compromising cancer cell proliferation.

    Journal: Oncotarget

    Article Title: WEE1 epigenetically modulates 5-hmC levels by pY37-H2B dependent regulation of IDH2 gene expression

    doi: 10.18632/oncotarget.22374

    Figure Lengend Snippet: WEE1 pY37-H2B epigenetic signaling axis downregtulates IDH2 mRNA expression and 5-hmC levels in cancer cell; a model WEE1 overexpressing cancer cells deposits pY37-H2B repressive marks to suppress the expression of IDH2 transcription which is reflected in a significant loss of global 5-hmC levels that promotes gene expression programs that favor tumor growth. Inhibition of WEE1 by AZD1775 led to the loss of pY37-H2B which in turn restored 5-hmC levels, compromising cancer cell proliferation.

    Article Snippet: WEE1, Actin and H2B antibodies were purchased from Santacruz.

    Techniques: Expressing, Inhibition

    Deposition of WEE1 mediated pY37-H2B epigenetic marks within the IDH2 gene locus ( A ) Deposition of pY37-H2B epigenetic marks in the mouse Idh2 and human IDH2 locus are shown. The red boxes are the exons and yellow ovals are the sites of pY37-H2B deposition. ( B ) ChIP with pY37-H2B and IgG antibodies, followed by qPCR with primers corresponding to the pY37-H2B binding site of mouse embryo fibroblasts (MEFs). Data represent mean ± s.e.m. ( n = 3. * p

    Journal: Oncotarget

    Article Title: WEE1 epigenetically modulates 5-hmC levels by pY37-H2B dependent regulation of IDH2 gene expression

    doi: 10.18632/oncotarget.22374

    Figure Lengend Snippet: Deposition of WEE1 mediated pY37-H2B epigenetic marks within the IDH2 gene locus ( A ) Deposition of pY37-H2B epigenetic marks in the mouse Idh2 and human IDH2 locus are shown. The red boxes are the exons and yellow ovals are the sites of pY37-H2B deposition. ( B ) ChIP with pY37-H2B and IgG antibodies, followed by qPCR with primers corresponding to the pY37-H2B binding site of mouse embryo fibroblasts (MEFs). Data represent mean ± s.e.m. ( n = 3. * p

    Article Snippet: WEE1, Actin and H2B antibodies were purchased from Santacruz.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay

    WEE1 inhibition suppresses pY37-H2B expression ( A – D ) Prostate cancer (LAPC4 and LNCaP), melanoma (WM1366) and brain cancer (T98G) derived cells were treated with AZD1775 (2 µM, 24 hr) and lysates were immunoprecipitated with pY37-H2B antibodies, followed by immunoblotting with pan-H2B antibodies (top panels). CL indicates the crude lysates. Lower panel indicates immunoblotting of lysates with actin antibodies. ( E ) Cell viability assay. Melanoma, prostate and brain cancer cell lines were treated with AZD1775 for 96 hr and live/dead population was assessed by Trypan blue staining. Data represent mean ± standard error of the mean or s.e.m. ( n = 3). ( F ) Table indicates IC 50 values based on cell proliferation analysis in AZD1775 treated cells in comparison to untreated cells shown in Figure 1E .

    Journal: Oncotarget

    Article Title: WEE1 epigenetically modulates 5-hmC levels by pY37-H2B dependent regulation of IDH2 gene expression

    doi: 10.18632/oncotarget.22374

    Figure Lengend Snippet: WEE1 inhibition suppresses pY37-H2B expression ( A – D ) Prostate cancer (LAPC4 and LNCaP), melanoma (WM1366) and brain cancer (T98G) derived cells were treated with AZD1775 (2 µM, 24 hr) and lysates were immunoprecipitated with pY37-H2B antibodies, followed by immunoblotting with pan-H2B antibodies (top panels). CL indicates the crude lysates. Lower panel indicates immunoblotting of lysates with actin antibodies. ( E ) Cell viability assay. Melanoma, prostate and brain cancer cell lines were treated with AZD1775 for 96 hr and live/dead population was assessed by Trypan blue staining. Data represent mean ± standard error of the mean or s.e.m. ( n = 3). ( F ) Table indicates IC 50 values based on cell proliferation analysis in AZD1775 treated cells in comparison to untreated cells shown in Figure 1E .

    Article Snippet: WEE1, Actin and H2B antibodies were purchased from Santacruz.

    Techniques: Inhibition, Expressing, Derivative Assay, Immunoprecipitation, Viability Assay, Staining

    Polyglutamine expansion of ATXN7 alters chromatin occupancy and histone H2B monoubiquitination at the RELN promoter and increases global H2B monoubiquitination. ( A ) Polyglutamine expansion of ATXN7 resulted in a reduction of polyQ ATXN7 occupancy at the

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

    Article Title: Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes

    doi: 10.1073/pnas.1218331110

    Figure Lengend Snippet: Polyglutamine expansion of ATXN7 alters chromatin occupancy and histone H2B monoubiquitination at the RELN promoter and increases global H2B monoubiquitination. ( A ) Polyglutamine expansion of ATXN7 resulted in a reduction of polyQ ATXN7 occupancy at the

    Article Snippet: Anti-Flag (no. F3156, Western blot; no. F1804, immunostaining and ChIP; Sigma), anti-GCN5 (no. 607202; Biolegend), anti-histone H2B (no. 17-10054; Millipore), anti-histone H2B ubiquitinated on lysine 120 (no.17-650; Millipore), anti-histone H3 (no. 39163; Active Motif), anti-histone H3 acetylated on lysine 9/14 (no. 06-599; Millipore), anti-histone H4 acetylated on lysine 16 (no. 39167; Active Motif), or anti-reelin (no. ab78540; Abcam).

    Techniques:

    Treatment with 200 ng/mL TSA alters ATXN7 protein levels, NI dynamics, and H2B monoubiquitination levels at the RELN promoter. ( A ) Levels of Flag-polyQ ATXN7 protein were increased in SCA7 astrocytes following 18 h of exposure to 200 ng/mL TSA as shown

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

    Article Title: Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes

    doi: 10.1073/pnas.1218331110

    Figure Lengend Snippet: Treatment with 200 ng/mL TSA alters ATXN7 protein levels, NI dynamics, and H2B monoubiquitination levels at the RELN promoter. ( A ) Levels of Flag-polyQ ATXN7 protein were increased in SCA7 astrocytes following 18 h of exposure to 200 ng/mL TSA as shown

    Article Snippet: Anti-Flag (no. F3156, Western blot; no. F1804, immunostaining and ChIP; Sigma), anti-GCN5 (no. 607202; Biolegend), anti-histone H2B (no. 17-10054; Millipore), anti-histone H2B ubiquitinated on lysine 120 (no.17-650; Millipore), anti-histone H3 (no. 39163; Active Motif), anti-histone H3 acetylated on lysine 9/14 (no. 06-599; Millipore), anti-histone H4 acetylated on lysine 16 (no. 39167; Active Motif), or anti-reelin (no. ab78540; Abcam).

    Techniques:

    Split GFP complementation assay to determine in-vivo association of LANA with histones. A. Schematic of split GFP fusion proteins and their association. B. Histone H1 or H2B fused with GFP-N term (1–157 aa) were co-transfected with LANA1–32 aa (top panel), LANA 1–340 aa (second panel), LANA-FL with wt CBD (third panel from top) and LANA-FL with alanine substituted 5–15 aa of CBD (bottom panel) fused with C-term of GFP (158–238 aa). GFP fluorescence was imaged after 48 h post- transfection. C. Western blot with anti-GFP antibody to show relatively similar expression of GFP-N term and C-term fused proteins in cells used for comparison of fluorescence. Band of interests are indicated with red arrow.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Split GFP complementation assay to determine in-vivo association of LANA with histones. A. Schematic of split GFP fusion proteins and their association. B. Histone H1 or H2B fused with GFP-N term (1–157 aa) were co-transfected with LANA1–32 aa (top panel), LANA 1–340 aa (second panel), LANA-FL with wt CBD (third panel from top) and LANA-FL with alanine substituted 5–15 aa of CBD (bottom panel) fused with C-term of GFP (158–238 aa). GFP fluorescence was imaged after 48 h post- transfection. C. Western blot with anti-GFP antibody to show relatively similar expression of GFP-N term and C-term fused proteins in cells used for comparison of fluorescence. Band of interests are indicated with red arrow.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: In Vivo, Transfection, Fluorescence, Western Blot, Expressing

    RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Stable Transfection, Expressing, Staining, Transfection, Immunoprecipitation, Western Blot

    Immune localization of LANA and histones in PEL cells. In the ‘Intact Cells’ panel, KSHV infected, BCBL1 and JSC1 cells were fixed on a glass cover slip before permeabilization with TritonX-100. Histone H1 and H2B were detected with mouse anti-H1 and Rabbit anti-H2B antibodies. LANA was detected with rat anti-LANA monoclonal antibody. Histone H1 and H2B were detected with AlexaFluor 488 (green) and LANA was detected with AlexaFluor 594 (red). Nuclei were stained with DAPI shown in blue. Image including DIC were captured using Olympus confocal microscope. In the ‘Chromosome Spreads’ panel, BCBL1 and JSC1 cells were treated with hypotonic solution to prepare chromosome spreads. These spreads on glass slides were fixed and permeabilized with TritonX-100. Histone H1 and H2B were stained with specific antibodies and were counter stained with AlexaFluor 488 (green) and LANA were detected with AlexaFluor 594 (red). Yellow signals in the merge panels show colocalization.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Immune localization of LANA and histones in PEL cells. In the ‘Intact Cells’ panel, KSHV infected, BCBL1 and JSC1 cells were fixed on a glass cover slip before permeabilization with TritonX-100. Histone H1 and H2B were detected with mouse anti-H1 and Rabbit anti-H2B antibodies. LANA was detected with rat anti-LANA monoclonal antibody. Histone H1 and H2B were detected with AlexaFluor 488 (green) and LANA was detected with AlexaFluor 594 (red). Nuclei were stained with DAPI shown in blue. Image including DIC were captured using Olympus confocal microscope. In the ‘Chromosome Spreads’ panel, BCBL1 and JSC1 cells were treated with hypotonic solution to prepare chromosome spreads. These spreads on glass slides were fixed and permeabilized with TritonX-100. Histone H1 and H2B were stained with specific antibodies and were counter stained with AlexaFluor 488 (green) and LANA were detected with AlexaFluor 594 (red). Yellow signals in the merge panels show colocalization.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Infection, Staining, Microscopy

    Histone H1 variants associated with LANA. H1 variants were cloned into a Flag epitope vector and transfected with a vector containing LANA-myc. Histone H2B-Flag was also transfected for comparison. Anti-myc IP to precipitate LANA co-precipitated H1.c as well as H1.x, the binding affinity of H1.c relatively stronger then H1.x. LANA in the input and IP lanes were detected in anti-myc blot.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Histone H1 variants associated with LANA. H1 variants were cloned into a Flag epitope vector and transfected with a vector containing LANA-myc. Histone H2B-Flag was also transfected for comparison. Anti-myc IP to precipitate LANA co-precipitated H1.c as well as H1.x, the binding affinity of H1.c relatively stronger then H1.x. LANA in the input and IP lanes were detected in anti-myc blot.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Clone Assay, FLAG-tag, Plasmid Preparation, Transfection, Binding Assay

    In-vitro binding of LANA with histones, H1 and H2B. A. Amino acid sequence of LANA 1–32 aa with CBD sequences in yellow. Alanine substitution mutants of CBD are shown in yellow. B. In-vitro translated histone H1 or H2B were incubated with GST (control) or GST-LANA1–32 and its alanine substitution mutants. Bound fraction of H1 and H2B were detected by 35S methionine label on H1 and H2B. Amounts of GST and LANA1–32 aa GST-fusion proteins used in the binding assay are shown in respective GST-protein panels. C. Histone H1 and H2B expressed in 293T cells were subjected for binding with GST (control), GST-LANA1–32 aa and CBD mutants. Bound histones were detected in a western blot with anti-HA antibody. GST proteins used in the binding assay are shown in GST-panels. D. LANA schematic showing the regions of LANA used as N-term (1–340 aa) and C-term (940–1162 aa). E. 35 S methionine labeled histone H1 and H2B were in-vitro translated for binding with GST (control), GST-LANA-N and GST-LANA-C fusion proteins. Relative densities (RD) of bound histone H1 (black bar) and H2B (white bar) with LANA-N and LANA-C were determined by comparing with respective inputs at 10%. F. In-vitro translated histone H1 and H2B were mixed before binding with GST fusion proteins. Relative binding of both the histones with LANA-N and C were determined by the densities of the bands in respective lanes. Commassie stained gel of GST-LANA-N and GST-LANA-C used in the assay. G . Schematic to show GST (control) and GST-H1 and GST-H2B fusion proteins. H . Schematic of LANA-FL with wt CBD (1) and its alanine mutants from 2–6. I. LANA and its alanine substitution mutants were in-vitro translated with 35 S methionine for binding with histone H1 and H2B. In-vitro translated products were subjected for binding with GST (control), GST-H1 and GST-H2B. The bound fractions were determined, which showed efficient binding of wt LANA as well as alanine mutants to histones, H1 and H2B. J. LANA with wt CBD and its alanine substitution mutants were expressed in 293T cells. Lysates were subjected for binding with GST (control), GST-H1 and GST-H2B. Bound fraction were detected with anti-myc WB, which showed binding of wt LANA as well as its mutants to both histone H1 and H2B but not to control GST.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: In-vitro binding of LANA with histones, H1 and H2B. A. Amino acid sequence of LANA 1–32 aa with CBD sequences in yellow. Alanine substitution mutants of CBD are shown in yellow. B. In-vitro translated histone H1 or H2B were incubated with GST (control) or GST-LANA1–32 and its alanine substitution mutants. Bound fraction of H1 and H2B were detected by 35S methionine label on H1 and H2B. Amounts of GST and LANA1–32 aa GST-fusion proteins used in the binding assay are shown in respective GST-protein panels. C. Histone H1 and H2B expressed in 293T cells were subjected for binding with GST (control), GST-LANA1–32 aa and CBD mutants. Bound histones were detected in a western blot with anti-HA antibody. GST proteins used in the binding assay are shown in GST-panels. D. LANA schematic showing the regions of LANA used as N-term (1–340 aa) and C-term (940–1162 aa). E. 35 S methionine labeled histone H1 and H2B were in-vitro translated for binding with GST (control), GST-LANA-N and GST-LANA-C fusion proteins. Relative densities (RD) of bound histone H1 (black bar) and H2B (white bar) with LANA-N and LANA-C were determined by comparing with respective inputs at 10%. F. In-vitro translated histone H1 and H2B were mixed before binding with GST fusion proteins. Relative binding of both the histones with LANA-N and C were determined by the densities of the bands in respective lanes. Commassie stained gel of GST-LANA-N and GST-LANA-C used in the assay. G . Schematic to show GST (control) and GST-H1 and GST-H2B fusion proteins. H . Schematic of LANA-FL with wt CBD (1) and its alanine mutants from 2–6. I. LANA and its alanine substitution mutants were in-vitro translated with 35 S methionine for binding with histone H1 and H2B. In-vitro translated products were subjected for binding with GST (control), GST-H1 and GST-H2B. The bound fractions were determined, which showed efficient binding of wt LANA as well as alanine mutants to histones, H1 and H2B. J. LANA with wt CBD and its alanine substitution mutants were expressed in 293T cells. Lysates were subjected for binding with GST (control), GST-H1 and GST-H2B. Bound fraction were detected with anti-myc WB, which showed binding of wt LANA as well as its mutants to both histone H1 and H2B but not to control GST.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Western Blot, Labeling, Staining

    The LANA-FL and 1–340 aa polypeptide precipitated histone H1 as well as H2B. A. Schematic of LANA 1–340 aa with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. B. HA tagged histone H1 or H2B was co-transfected with myc vector (Vec) or LANA 1–340 aa-myc (lane 1) and its CBD mutants (lanes 2–6). Lane M was the marker and lane Vec contain HA-H1 or H2B co-transfected with myc vector. LANA1–340 aa and its alanine mutants were immunoprecipitated with anti-myc antibody and detected with myc antibody (WB:myc). Histones, H1 and H2B in the input and IP lanes were detected with anti-HA antibody (WB:HA). LANA1–340 aa with wt CBD (lane 1) co-precipitated histone H1 and a small amount with CBD mutant 14-TG-15 (lane 5). Histone H2B was co-precipitated with LANA 1–340 aa with wt CBD (lane 1) as well as alanine mutants in lane 3, 4, 5 and small amount in lane 6. C. HA tagged histone H1 or H2B was co-transfected with myc vector (Vec) or LANA-FL-myc (lane 1) and its CBD mutants (lanes 2–6). LANA and its alanine mutants were immunoprecipitated with anti-myc antibody and detected with myc antibody (WB:myc). Histones, H1 and H2B in the input and IP lanes were detected with anti-HA antibody (WB:HA). LANA with wt CBD (lane 1) as well as all the alanine mutants (lanes, 2–6) co-precipitated histone H1. Histone H2B was co-precipitated with LANA containing wt CBD and all the alanine substitution mutants (lanes 2–6).

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: The LANA-FL and 1–340 aa polypeptide precipitated histone H1 as well as H2B. A. Schematic of LANA 1–340 aa with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. B. HA tagged histone H1 or H2B was co-transfected with myc vector (Vec) or LANA 1–340 aa-myc (lane 1) and its CBD mutants (lanes 2–6). Lane M was the marker and lane Vec contain HA-H1 or H2B co-transfected with myc vector. LANA1–340 aa and its alanine mutants were immunoprecipitated with anti-myc antibody and detected with myc antibody (WB:myc). Histones, H1 and H2B in the input and IP lanes were detected with anti-HA antibody (WB:HA). LANA1–340 aa with wt CBD (lane 1) co-precipitated histone H1 and a small amount with CBD mutant 14-TG-15 (lane 5). Histone H2B was co-precipitated with LANA 1–340 aa with wt CBD (lane 1) as well as alanine mutants in lane 3, 4, 5 and small amount in lane 6. C. HA tagged histone H1 or H2B was co-transfected with myc vector (Vec) or LANA-FL-myc (lane 1) and its CBD mutants (lanes 2–6). LANA and its alanine mutants were immunoprecipitated with anti-myc antibody and detected with myc antibody (WB:myc). Histones, H1 and H2B in the input and IP lanes were detected with anti-HA antibody (WB:HA). LANA with wt CBD (lane 1) as well as all the alanine mutants (lanes, 2–6) co-precipitated histone H1. Histone H2B was co-precipitated with LANA containing wt CBD and all the alanine substitution mutants (lanes 2–6).

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Transfection, Plasmid Preparation, Marker, Immunoprecipitation, Western Blot, Mutagenesis

    Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Stable Transfection, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Staining, Transfection, Western Blot, Marker, Mutagenesis, Immunoprecipitation

    LANA associates with histones and non-histones nucleosomal proteins. A. HA tagged Histone H1, H2B, MeCP2, DEK, HMG-N1 and HMG-N2 were co-transfected with LANA-myc. LANA was precipitated with anti-myc antibody and co-precipitating proteins were detected with anti-HA blot (IB:HA). M is the marker lane. Co-immunoprecipitating proteins are indicated with red triangle. B. HA tagged MeCP2 and DEK together were transfected with myc vector or LANA-myc followed by anti-myc immunoprecipitation (IP). The complex were resolved to separate DEK from the heavy chain and immune detection with anti-HA confirmed binding of both MeCP2 and DEK to LANA. C. HA tagged MeCP2 was transfected with LANA-N or LANA-C (fused to GFP and myc) along with control GFP-NLS-myc followed by anti-myc immunoprecipitation. Detection of MeCP2 with anti-HA blot showed its binding with C-terms of LANA (IB:HA, red triangle). GFP-NLS-myc, GFP-LANA-N and GFP-LANA-C in the input and IP lanes are indicated by red triangle. D. Beta-galactosidase activity of LANA interacting proteins. Yeast 190 stable expressing LANA-N term fused to GAL4DBD was co-transformed with LANA interacting proteins (H1, H2B, MeCP2, DEK, HMG-N1 and HMG-N2) fused to ACT domain of GAL4. Induction of beta-galactosidase due to the interaction of LANA bait with prey was detected by ONPG assay. Relative activity normalized against LANA-N fused to GAL4DBD with vector GAL4ACT, are shown in the bar graph from three independent experiments with three independent clones. Standard error bars were calculated based on the data from three independent experiments. Luciferase fused GAL4ACT was used as a negative control, which showed no induction of beta-galactosidase activity.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: LANA associates with histones and non-histones nucleosomal proteins. A. HA tagged Histone H1, H2B, MeCP2, DEK, HMG-N1 and HMG-N2 were co-transfected with LANA-myc. LANA was precipitated with anti-myc antibody and co-precipitating proteins were detected with anti-HA blot (IB:HA). M is the marker lane. Co-immunoprecipitating proteins are indicated with red triangle. B. HA tagged MeCP2 and DEK together were transfected with myc vector or LANA-myc followed by anti-myc immunoprecipitation (IP). The complex were resolved to separate DEK from the heavy chain and immune detection with anti-HA confirmed binding of both MeCP2 and DEK to LANA. C. HA tagged MeCP2 was transfected with LANA-N or LANA-C (fused to GFP and myc) along with control GFP-NLS-myc followed by anti-myc immunoprecipitation. Detection of MeCP2 with anti-HA blot showed its binding with C-terms of LANA (IB:HA, red triangle). GFP-NLS-myc, GFP-LANA-N and GFP-LANA-C in the input and IP lanes are indicated by red triangle. D. Beta-galactosidase activity of LANA interacting proteins. Yeast 190 stable expressing LANA-N term fused to GAL4DBD was co-transformed with LANA interacting proteins (H1, H2B, MeCP2, DEK, HMG-N1 and HMG-N2) fused to ACT domain of GAL4. Induction of beta-galactosidase due to the interaction of LANA bait with prey was detected by ONPG assay. Relative activity normalized against LANA-N fused to GAL4DBD with vector GAL4ACT, are shown in the bar graph from three independent experiments with three independent clones. Standard error bars were calculated based on the data from three independent experiments. Luciferase fused GAL4ACT was used as a negative control, which showed no induction of beta-galactosidase activity.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Transfection, Marker, Plasmid Preparation, Immunoprecipitation, Binding Assay, Activity Assay, Expressing, Transformation Assay, Activated Clotting Time Assay, Clone Assay, Luciferase, Negative Control

    Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation

    Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Fluorescence, Förster Resonance Energy Transfer, Stable Transfection, Transduction, Transfection, Expressing, Software, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation

    LANA binds to histones H2B and histone H1. A. In-vitro translated LANA binds to purified histone H1 and H2B. Myc epitope tagged LANA was in-vitro translated and incubated with purified histone containing linker as well as core histones. Immunoprecipitation of LANA with anti-myc antibody and immunoblot (IB) using anti-histone H1 and anti-H2B showed binding with LANA (lane 3). Relative binding of histone H1 and H2B were determined by setting the intensity of input band (lane 1) at 10% and calculating the relative densities of based on band intensity of input lanes. Isotype control antibody did not show any delectable binding (lane 2). B. 30 millions BCBL1 cells were lysed in RIPA buffer to immunoprecipitate LANA using anti-LANA antibody and detection of co-precipitated histone H1 and H2B. Immunoblot (IB) with anti-H1 and anti-H2B showed co-immunoprecipitation of both the histones (lane 2). Isotype control antibody did not now any detectable binding (lane 3). Relative binding of histone H1 and H2B were determined by setting the densities of input lanes (lane 1) at 10% and calculating the relative densities of immunoprecipitated bands in lane 2. C. Lysates of BCBL1 from 30 million cells were treated with 2 U of micrococcal nuclease (MNase) to digest the inter-nucleosomal DNA for generating mononucleosomes. MNase treated lysates was subjected for immunoprecipitation with anti-LANA antibody. Immune detection of histone H2B showed reduced level of H2B as compared to H1 (lane 2). Relative density calculated by using input as 10% showed reduced binding of histone H1 (approx. 10%) as compared to 30% without MNase treatment.

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: LANA binds to histones H2B and histone H1. A. In-vitro translated LANA binds to purified histone H1 and H2B. Myc epitope tagged LANA was in-vitro translated and incubated with purified histone containing linker as well as core histones. Immunoprecipitation of LANA with anti-myc antibody and immunoblot (IB) using anti-histone H1 and anti-H2B showed binding with LANA (lane 3). Relative binding of histone H1 and H2B were determined by setting the intensity of input band (lane 1) at 10% and calculating the relative densities of based on band intensity of input lanes. Isotype control antibody did not show any delectable binding (lane 2). B. 30 millions BCBL1 cells were lysed in RIPA buffer to immunoprecipitate LANA using anti-LANA antibody and detection of co-precipitated histone H1 and H2B. Immunoblot (IB) with anti-H1 and anti-H2B showed co-immunoprecipitation of both the histones (lane 2). Isotype control antibody did not now any detectable binding (lane 3). Relative binding of histone H1 and H2B were determined by setting the densities of input lanes (lane 1) at 10% and calculating the relative densities of immunoprecipitated bands in lane 2. C. Lysates of BCBL1 from 30 million cells were treated with 2 U of micrococcal nuclease (MNase) to digest the inter-nucleosomal DNA for generating mononucleosomes. MNase treated lysates was subjected for immunoprecipitation with anti-LANA antibody. Immune detection of histone H2B showed reduced level of H2B as compared to H1 (lane 2). Relative density calculated by using input as 10% showed reduced binding of histone H1 (approx. 10%) as compared to 30% without MNase treatment.

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: In Vitro, Purification, Incubation, Immunoprecipitation, Binding Assay

    Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).

    Journal: PLoS ONE

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    doi: 10.1371/journal.pone.0074662

    Figure Lengend Snippet: Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).

    Article Snippet: Anti-histone H1 Antibody (clone AE-4) and Anti-histone H2B Antibody were purchased from Millipore (EMD Millipore Corporation).

    Techniques: Infection, Transduction, Expressing, Staining, Stable Transfection, Immunoprecipitation, Western Blot

    Histones are on mammalian LDs and respond to LPS. ( A ). Western blot analysis of LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS. Antibodies against ALDI, histones H2A, H2B and H3 were used. Whole liver homogenate (h) was used for comparison and as a control. ( B ) The presence of histone H1 (H1) on LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS was detected by immunoblot, and more H1 was present on droplets purified from LPS-treated animals. Equal total proteins from the nuclear fraction (n) and from whole liver homogenate (h) were used as comparison. ( C ). Mice were injected intraperitoneally with (+) or without (−) LPS, and transaminase levels (AST and ALT) and cytokine levels (IL-6 and TNFα) were quantified in units/l or units/ml in the serum; asterisk indicates statistical significance (p=0.05), confirming that LPS injection provoked the expected biological response. ( D ). Western blot analysis of histone H1 released into the buffer (UB) when purified LDs, from the liver of infection induced mice, were treated with LPS (+). Histone H1 is either minimally detected or not at all detected in the buffer in the absence of LPS (−). The band at 97 kDa in C and D represents histone H1 oligomers. DOI: http://dx.doi.org/10.7554/eLife.00003.011

    Journal: eLife

    Article Title: A novel role for lipid droplets in the organismal antibacterial responseDecision letterAuthor response

    doi: 10.7554/eLife.00003.013

    Figure Lengend Snippet: Histones are on mammalian LDs and respond to LPS. ( A ). Western blot analysis of LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS. Antibodies against ALDI, histones H2A, H2B and H3 were used. Whole liver homogenate (h) was used for comparison and as a control. ( B ) The presence of histone H1 (H1) on LDs (LD) purified from hepatocytes of mice injected with (+) or without (−) LPS was detected by immunoblot, and more H1 was present on droplets purified from LPS-treated animals. Equal total proteins from the nuclear fraction (n) and from whole liver homogenate (h) were used as comparison. ( C ). Mice were injected intraperitoneally with (+) or without (−) LPS, and transaminase levels (AST and ALT) and cytokine levels (IL-6 and TNFα) were quantified in units/l or units/ml in the serum; asterisk indicates statistical significance (p=0.05), confirming that LPS injection provoked the expected biological response. ( D ). Western blot analysis of histone H1 released into the buffer (UB) when purified LDs, from the liver of infection induced mice, were treated with LPS (+). Histone H1 is either minimally detected or not at all detected in the buffer in the absence of LPS (−). The band at 97 kDa in C and D represents histone H1 oligomers. DOI: http://dx.doi.org/10.7554/eLife.00003.011

    Article Snippet: The presence of proteins histones H2A and H2B, and kinesin heavy chain in droplets was monitored using immunoblot analysis with anti-H2A , anti-H2B (Upstate Biotechnologies, Lake Placid, NY, USA), and anti-Khc (Cytoskeleton, CO, USA), respectively.

    Techniques: Western Blot, Purification, Mouse Assay, Injection, AST Assay, Infection

    Presence of extranuclear histones depends on the Jabba protein. ( A ) Histone H2Av GFP is not detectable in cytoplasmic puncta of Jabba zl01 embryos. Both genotypes show strong signal in nuclei. ( B ). By immunostaining, endogenous H2A and H2B are absent from the lipid droplet layer (LD) of centrifuged Jabba zl01 embryos. ( C ). Histone H2A is absent from the lipid droplet layer in centrifuged Jabba f07560 embryos. BF is the bright field image and LD is the lipid droplet layer. ( D ). Equal amounts of proteins from purified LDs were compared by Western analysis. Droplets from Jabba zl01 embryos lack histones H2A and H2B. The droplet-bound Khc protein serves as loading control. ( E ). When compared side by side, similar reductions in droplet-bound histones were found for both the independently isolated Jabba alleles Jabba f07560 and Jabba zlo1 . ( F ). Western blot of equal numbers of unfertilized wild-type and Jabba mutant embryos. Overall levels of histone H2A and H2B are significantly reduced in the Jabba mutants. ( G ). LDs purified from embryos of two independently isolated Jabba mutants revealed no bacterial killing activity in antibacterial plate assays, with bacterial growth comparable to buffer alone, in contrast to droplets purified from wild-type embryos which dramatically decreased bacterial growth. DOI: http://dx.doi.org/10.7554/eLife.00003.004

    Journal: eLife

    Article Title: A novel role for lipid droplets in the organismal antibacterial responseDecision letterAuthor response

    doi: 10.7554/eLife.00003.013

    Figure Lengend Snippet: Presence of extranuclear histones depends on the Jabba protein. ( A ) Histone H2Av GFP is not detectable in cytoplasmic puncta of Jabba zl01 embryos. Both genotypes show strong signal in nuclei. ( B ). By immunostaining, endogenous H2A and H2B are absent from the lipid droplet layer (LD) of centrifuged Jabba zl01 embryos. ( C ). Histone H2A is absent from the lipid droplet layer in centrifuged Jabba f07560 embryos. BF is the bright field image and LD is the lipid droplet layer. ( D ). Equal amounts of proteins from purified LDs were compared by Western analysis. Droplets from Jabba zl01 embryos lack histones H2A and H2B. The droplet-bound Khc protein serves as loading control. ( E ). When compared side by side, similar reductions in droplet-bound histones were found for both the independently isolated Jabba alleles Jabba f07560 and Jabba zlo1 . ( F ). Western blot of equal numbers of unfertilized wild-type and Jabba mutant embryos. Overall levels of histone H2A and H2B are significantly reduced in the Jabba mutants. ( G ). LDs purified from embryos of two independently isolated Jabba mutants revealed no bacterial killing activity in antibacterial plate assays, with bacterial growth comparable to buffer alone, in contrast to droplets purified from wild-type embryos which dramatically decreased bacterial growth. DOI: http://dx.doi.org/10.7554/eLife.00003.004

    Article Snippet: The presence of proteins histones H2A and H2B, and kinesin heavy chain in droplets was monitored using immunoblot analysis with anti-H2A , anti-H2B (Upstate Biotechnologies, Lake Placid, NY, USA), and anti-Khc (Cytoskeleton, CO, USA), respectively.

    Techniques: Immunostaining, Purification, Western Blot, Isolation, Mutagenesis, Activity Assay

    Bacterial cell wall components release droplet bounds histones in a dose dependent manner. ( A ). Increasing concentrations of lipopolysaccharide (LPS) in the buffer releases droplet bound histones from purified LDs. ( B ). Lipoteichoic acid (LTA) causes the dose dependent release of histones from purified droplets. LDs were purified from wild-type Drosophila embryos, re-suspended in buffer, and incubated for 2 hr at room temperature with different concentrations of LPS or LTA. LDs (LD) were then separated from the under-layer buffer (UB) and both were processed for SDS-PAGE. Western Blot analysis was carried out with H2B histone antibodies. DOI: http://dx.doi.org/10.7554/eLife.00003.010

    Journal: eLife

    Article Title: A novel role for lipid droplets in the organismal antibacterial responseDecision letterAuthor response

    doi: 10.7554/eLife.00003.013

    Figure Lengend Snippet: Bacterial cell wall components release droplet bounds histones in a dose dependent manner. ( A ). Increasing concentrations of lipopolysaccharide (LPS) in the buffer releases droplet bound histones from purified LDs. ( B ). Lipoteichoic acid (LTA) causes the dose dependent release of histones from purified droplets. LDs were purified from wild-type Drosophila embryos, re-suspended in buffer, and incubated for 2 hr at room temperature with different concentrations of LPS or LTA. LDs (LD) were then separated from the under-layer buffer (UB) and both were processed for SDS-PAGE. Western Blot analysis was carried out with H2B histone antibodies. DOI: http://dx.doi.org/10.7554/eLife.00003.010

    Article Snippet: The presence of proteins histones H2A and H2B, and kinesin heavy chain in droplets was monitored using immunoblot analysis with anti-H2A , anti-H2B (Upstate Biotechnologies, Lake Placid, NY, USA), and anti-Khc (Cytoskeleton, CO, USA), respectively.

    Techniques: Purification, Incubation, SDS Page, Western Blot

    The Jabba protein contributes to improved survival for adult flies. ( A and B ) Adult Drosophila lacking Jabba ( A : Jabba f07560 ; B : Jabba zl01 ) have reduced survival when challenged by bacteria. Wild-type and Jabba mutant ( Jabba zl0 and Jabba f07560 ) adult flies were infected with Listeria monocytogenes as detailed in ‘Materials and methods’, and fly survival was monitored over the course of 4 days. ( C ). Representative plates in a colony forming assay, showing bacterial colonies on agar plates streaked with cytosolic extract from bacteria infected adult flies. ( D ) Western blots of histone H2B from equal amounts of cytosolic extracts from wild type and Jabba mutant adult flies, showing that overall levels of H2B were significantly reduced in the Jabba mutants. DOI: http://dx.doi.org/10.7554/eLife.00003.009

    Journal: eLife

    Article Title: A novel role for lipid droplets in the organismal antibacterial responseDecision letterAuthor response

    doi: 10.7554/eLife.00003.013

    Figure Lengend Snippet: The Jabba protein contributes to improved survival for adult flies. ( A and B ) Adult Drosophila lacking Jabba ( A : Jabba f07560 ; B : Jabba zl01 ) have reduced survival when challenged by bacteria. Wild-type and Jabba mutant ( Jabba zl0 and Jabba f07560 ) adult flies were infected with Listeria monocytogenes as detailed in ‘Materials and methods’, and fly survival was monitored over the course of 4 days. ( C ). Representative plates in a colony forming assay, showing bacterial colonies on agar plates streaked with cytosolic extract from bacteria infected adult flies. ( D ) Western blots of histone H2B from equal amounts of cytosolic extracts from wild type and Jabba mutant adult flies, showing that overall levels of H2B were significantly reduced in the Jabba mutants. DOI: http://dx.doi.org/10.7554/eLife.00003.009

    Article Snippet: The presence of proteins histones H2A and H2B, and kinesin heavy chain in droplets was monitored using immunoblot analysis with anti-H2A , anti-H2B (Upstate Biotechnologies, Lake Placid, NY, USA), and anti-Khc (Cytoskeleton, CO, USA), respectively.

    Techniques: Mutagenesis, Infection, Western Blot

    LDs kill bacteria via droplet bound histones. ( A ). Representative plates in a colony forming assay, showing growth of Gram-negative ( Escherichia coli DH5α , top) and Gram-positive ( Staphylococcus epidermidis , bottom) bacteria, where a known amount of bacteria were incubated at 37°C either in buffer alone, or with LDs pre-treated with or without anti-histone antibodies. In buffer (left, ‘Buffer’), many colonies (white spots) were observed, but in the presence of LDs (LD), the observed number of colonies was greatly decreased, demonstrating an antibacterial effect of the LDs. Pre-treatment of the droplets with anti-histone antibodies abolished this effect (LD + Anti-histones). ( B ). Quantification of colony forming assay in A . Each bar represents the mean number of observed colonies, in three independent trials, presented with the standard error. ( C ). Disc diffusion assay over a lawn of E. coli DH5α. A potential antibacterial agent is placed on a small sterile piece of filter paper (white circle); a cleared area (darker region) indicates antibacterial activity. Positive control: the antibiotic kanamycin (Antibiotic); growth inhibition region indicated by the red arrow. Negative control: buffer (Buffer). LDs isolated in the presence (LD-CaCO 3 ) or absence (LD) of alkaline carbonate were spotted on sterile discs; bacterial inhibition was observed in the untreated droplets (LD) but not in the carbonate-treated droplets (LD-CaCO 3 ). The filter papers are 7 mm in diameter. ( D ). Quantification of the size of the clear zone in the disc diffusion assay from C . Fifteen independent disc diffusion assays were performed with purified LDs from Drosophila embryos, the antibiotic kanamycin, or buffer. Antimicrobial activity of compounds was quantified as the diameter of the clear zones surrounding the filter papers after subtraction of filter papers diameter (1 A.U. = 0.1 mm). ( E ). Use of a gel-overlay assay to determine the identity of the anti-bacterial protein(s) on the LDs. Proteins extracted from LDs (LD, left lane) were run in duplicate on an AU-gel; murine cryptdin 4 (Crp 4, right lane) served as positive control. After electrophoresis, the gel was split. One half (left) was stained by Coomassie Blue; the histone bands are indicated by a blue arrow and the crp 4 control is indicated by the green arrow. The other half (right) was used in a gel overlay assay (see ‘Materials and methods’) to reveal regions of the gel able to inhibit bacterial growth (inhibition by LD is indicated by the red arrow and that by crp 4 control is indicated by the violet arrow). Inhibition of bacterial growth due to proteins on the LDs was only observed in a single region, corresponding to the histones (red arrow). Consistent with this, mass spectrometry of proteins cut from the Coomassie gel corresponding to the killing region identified predominantly histones H2A and H2B (see ‘LDs have antimicrobial activity’). ( F ). E. coli ML35 cultures were transiently (∼1 hr) exposed to commercial calf thymus pan-histone proteins (Sigma) or gel-extracted LD-histones (from the gel-overlay assay). Both preparations show similar potency for bacterial killing. DOI: http://dx.doi.org/10.7554/eLife.00003.003

    Journal: eLife

    Article Title: A novel role for lipid droplets in the organismal antibacterial responseDecision letterAuthor response

    doi: 10.7554/eLife.00003.013

    Figure Lengend Snippet: LDs kill bacteria via droplet bound histones. ( A ). Representative plates in a colony forming assay, showing growth of Gram-negative ( Escherichia coli DH5α , top) and Gram-positive ( Staphylococcus epidermidis , bottom) bacteria, where a known amount of bacteria were incubated at 37°C either in buffer alone, or with LDs pre-treated with or without anti-histone antibodies. In buffer (left, ‘Buffer’), many colonies (white spots) were observed, but in the presence of LDs (LD), the observed number of colonies was greatly decreased, demonstrating an antibacterial effect of the LDs. Pre-treatment of the droplets with anti-histone antibodies abolished this effect (LD + Anti-histones). ( B ). Quantification of colony forming assay in A . Each bar represents the mean number of observed colonies, in three independent trials, presented with the standard error. ( C ). Disc diffusion assay over a lawn of E. coli DH5α. A potential antibacterial agent is placed on a small sterile piece of filter paper (white circle); a cleared area (darker region) indicates antibacterial activity. Positive control: the antibiotic kanamycin (Antibiotic); growth inhibition region indicated by the red arrow. Negative control: buffer (Buffer). LDs isolated in the presence (LD-CaCO 3 ) or absence (LD) of alkaline carbonate were spotted on sterile discs; bacterial inhibition was observed in the untreated droplets (LD) but not in the carbonate-treated droplets (LD-CaCO 3 ). The filter papers are 7 mm in diameter. ( D ). Quantification of the size of the clear zone in the disc diffusion assay from C . Fifteen independent disc diffusion assays were performed with purified LDs from Drosophila embryos, the antibiotic kanamycin, or buffer. Antimicrobial activity of compounds was quantified as the diameter of the clear zones surrounding the filter papers after subtraction of filter papers diameter (1 A.U. = 0.1 mm). ( E ). Use of a gel-overlay assay to determine the identity of the anti-bacterial protein(s) on the LDs. Proteins extracted from LDs (LD, left lane) were run in duplicate on an AU-gel; murine cryptdin 4 (Crp 4, right lane) served as positive control. After electrophoresis, the gel was split. One half (left) was stained by Coomassie Blue; the histone bands are indicated by a blue arrow and the crp 4 control is indicated by the green arrow. The other half (right) was used in a gel overlay assay (see ‘Materials and methods’) to reveal regions of the gel able to inhibit bacterial growth (inhibition by LD is indicated by the red arrow and that by crp 4 control is indicated by the violet arrow). Inhibition of bacterial growth due to proteins on the LDs was only observed in a single region, corresponding to the histones (red arrow). Consistent with this, mass spectrometry of proteins cut from the Coomassie gel corresponding to the killing region identified predominantly histones H2A and H2B (see ‘LDs have antimicrobial activity’). ( F ). E. coli ML35 cultures were transiently (∼1 hr) exposed to commercial calf thymus pan-histone proteins (Sigma) or gel-extracted LD-histones (from the gel-overlay assay). Both preparations show similar potency for bacterial killing. DOI: http://dx.doi.org/10.7554/eLife.00003.003

    Article Snippet: The presence of proteins histones H2A and H2B, and kinesin heavy chain in droplets was monitored using immunoblot analysis with anti-H2A , anti-H2B (Upstate Biotechnologies, Lake Placid, NY, USA), and anti-Khc (Cytoskeleton, CO, USA), respectively.

    Techniques: Incubation, Diffusion-based Assay, Activity Assay, Positive Control, Inhibition, Negative Control, Isolation, Purification, Overlay Assay, Electrophoresis, Staining, Mass Spectrometry

    Representative images, size distribution and fluorescence signal intensity from fluorescence marker of canonical histones (H2A, H2B, H3, H4) and 2 large variants of histone H2A (macroH2A1.1 and macroH2A1.2). ImageStream photographs show bright-field images and histone staining (fluorescence from Alexa Fluor® 488, Alexa Fluor® 549, Alexa Fluor® 594 or Alexa Fluor® 647)

    Journal: Clinical Epigenetics

    Article Title: Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD)

    doi: 10.1186/s13148-020-00917-2

    Figure Lengend Snippet: Representative images, size distribution and fluorescence signal intensity from fluorescence marker of canonical histones (H2A, H2B, H3, H4) and 2 large variants of histone H2A (macroH2A1.1 and macroH2A1.2). ImageStream photographs show bright-field images and histone staining (fluorescence from Alexa Fluor® 488, Alexa Fluor® 549, Alexa Fluor® 594 or Alexa Fluor® 647)

    Article Snippet: Primary antibodies: anti-mH2A (Abcam, Ab18255, USA), anti-histone H2B (Abcam, Ab134211, USA), anti-histone H4 (Abcam, Ab31830, USA), anti-histone H3 (Abcam, Ab12079, USA).

    Techniques: Fluorescence, Marker, Staining

    Representative images, size distribution and fluorescence signal intensity from Alexa Fluor® 488 and Alexa Fluor® 594 of multi-channel detecting of macroH2A1.2/H2B histone. ImageStream photographs show bright-field images, macroH2A1.2 histone staining (fluorescence from Alexa Fluor® 488), H2B histone staining (fluorescence from Alexa Fluor® 594) and composite images, overlays of macroH2A1.2 and H2B staining.

    Journal: Clinical Epigenetics

    Article Title: Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD)

    doi: 10.1186/s13148-020-00917-2

    Figure Lengend Snippet: Representative images, size distribution and fluorescence signal intensity from Alexa Fluor® 488 and Alexa Fluor® 594 of multi-channel detecting of macroH2A1.2/H2B histone. ImageStream photographs show bright-field images, macroH2A1.2 histone staining (fluorescence from Alexa Fluor® 488), H2B histone staining (fluorescence from Alexa Fluor® 594) and composite images, overlays of macroH2A1.2 and H2B staining.

    Article Snippet: Primary antibodies: anti-mH2A (Abcam, Ab18255, USA), anti-histone H2B (Abcam, Ab134211, USA), anti-histone H4 (Abcam, Ab31830, USA), anti-histone H3 (Abcam, Ab12079, USA).

    Techniques: Fluorescence, Staining

    MK-4 inhibits rotenone-induced nuclear accumulation of the NF-κB p65 subunit in BV2 cells. (A) Various doses of MK-4 and 1 μmol/L rotenone were administered to BV2 cells as indicated for 6 h. The cells were fixed and labeled with anti-p65 (red) antibodies. Then, the cells were visualized under a fluorescent microscope. The nuclei were stained with DAPI (1 μg/mL) (blue). Scale bars, 5 μm. (B) The cytoplasmic and nuclear fractions from the treated BV2 cells were immunoblotted with anti-p65, anti-GAPDH or anti-histone 2B antibodies. The band intensities of p65 relative to that of GAPDH (cytoplasmic fraction) or histone 2B (nuclear fraction) are shown in the lower two panels. The value of the group without drug (dissolvent only) is normalized as 1. The data are presented as the mean±SEM from three independent experiments. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Vitamin K2 suppresses rotenone-induced microglial activation in vitro

    doi: 10.1038/aps.2016.68

    Figure Lengend Snippet: MK-4 inhibits rotenone-induced nuclear accumulation of the NF-κB p65 subunit in BV2 cells. (A) Various doses of MK-4 and 1 μmol/L rotenone were administered to BV2 cells as indicated for 6 h. The cells were fixed and labeled with anti-p65 (red) antibodies. Then, the cells were visualized under a fluorescent microscope. The nuclei were stained with DAPI (1 μg/mL) (blue). Scale bars, 5 μm. (B) The cytoplasmic and nuclear fractions from the treated BV2 cells were immunoblotted with anti-p65, anti-GAPDH or anti-histone 2B antibodies. The band intensities of p65 relative to that of GAPDH (cytoplasmic fraction) or histone 2B (nuclear fraction) are shown in the lower two panels. The value of the group without drug (dissolvent only) is normalized as 1. The data are presented as the mean±SEM from three independent experiments. * P

    Article Snippet: An immunoblot analysis was performed using the following primary antibodies: monoclonal anti-GAPDH antibody was from Millipore; monoclonal anti-p38 antibody was from Santa Cruz Biotechnology (Dallas, Texas, USA); polyclonal anti-iNOS antibodies were from ABCAM (Cambridge, UK); polyclonal anti-COX2, anti-histone 2B, anti-IκB, and anti-p65 antibodies were from Epitomics (Cambridge, UK); polyclonal anti-IKK, anti-p-IKK (176/180), and anti-p-p38 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Labeling, Microscopy, Staining

    Histones lost from LPS-challenged BMDMs are released into the medium, in soluble and EV-associated forms. (A) The supernatant of BMDMs challenged with LPS for 4 h was collected, centrifuged to eliminate cell debris, and then large vesicles that were retrieved separately [macrobodies (MBs)]. The concentrated cell-conditioned medium (CCM) was then loaded onto a discontinuous Optiprep density gradient. Twelve fractions were collected, and an aliquot was tested by Western blotting for the presence of histone H3; the remainder was centrifuged, and the pellets were dissolved in hot SDS-PAGE loading buffer. Microvesicles and exosomes (MEs)-containing fractions were identified by the presence of TSG101, a membrane marker ( 19 ). One representative experiment, of four performed, is shown. (B) Extracellular vesicles in supernatants of H2B-GFP-expressing BMDMs were trapped by beads coated with anti-CD63 antibodies; beads were analyzed for GFP fluorescence by flow cytometry. MEs contain distinctly more H2B-GFP than macrobodies ( n = 3, t -test, * p

    Journal: Frontiers in Immunology

    Article Title: LPS-Challenged Macrophages Release Microvesicles Coated With Histones

    doi: 10.3389/fimmu.2018.01463

    Figure Lengend Snippet: Histones lost from LPS-challenged BMDMs are released into the medium, in soluble and EV-associated forms. (A) The supernatant of BMDMs challenged with LPS for 4 h was collected, centrifuged to eliminate cell debris, and then large vesicles that were retrieved separately [macrobodies (MBs)]. The concentrated cell-conditioned medium (CCM) was then loaded onto a discontinuous Optiprep density gradient. Twelve fractions were collected, and an aliquot was tested by Western blotting for the presence of histone H3; the remainder was centrifuged, and the pellets were dissolved in hot SDS-PAGE loading buffer. Microvesicles and exosomes (MEs)-containing fractions were identified by the presence of TSG101, a membrane marker ( 19 ). One representative experiment, of four performed, is shown. (B) Extracellular vesicles in supernatants of H2B-GFP-expressing BMDMs were trapped by beads coated with anti-CD63 antibodies; beads were analyzed for GFP fluorescence by flow cytometry. MEs contain distinctly more H2B-GFP than macrobodies ( n = 3, t -test, * p

    Article Snippet: Blocked membranes were probed with the following antibodies in TBS-T at 4°C for 16–24 h: rabbit Chip-Grade anti-H3 antibody (1:1,000; ab1791, Abcam plc, Cambridge, UK), anti-H2A (1:1,000; ab18255, Abcam plc, Cambridge, UK), anti-H2B (1:250; Upstate #07-371, Merck Millipore, KGaA, Darmstadt, Germany), mouse anti-beta-actin (1:2,000; A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-flotillin (1:250; #610820, BD Bioscience, San Jose, CA, USA), anti-TSG101 (1:1,000; ab30871, Abcam plc, Cambridge, UK), anti-citrullinated H3 (1:1,000; ab5103, Abcam plc, Cambridge, UK), anti-HLA IA/IE-PE (BD PharmingenTM).

    Techniques: Western Blot, SDS Page, Marker, Expressing, Fluorescence, Flow Cytometry, Cytometry

    Histones are present on the outer surface of microvesicles and exosomes (MEs). (A) H2B-GFP mice were exposed to doxycycline to induce H2B-GFP expression. Three weeks later, the mice were sacrificed and their bone marrows were harvested and cultured without doxycycline. In this way, bone marrow-derived macrophages (BMDMs) only contained H2B-GFP synthesized several weeks earlier, and all H2B-GFP was chromatin-associated. H2B-GFP BMDMs were then challenged with LPS, and imaged by stochastic optical reconstruction microscopy. H2B-GFP is green; cell membranes were stained red with concanavalin A-TRITC. Zooms of the regions inside the white squares are shown. (B) Western blot of macrobodies and MEs treated with trypsin in the presence or not of detergent (Triton X-100). The electron micrographs show representative MEs before and after trypsinization. (C) Transmission electron microscopy images of exosomes from LPS-challenged BMDMs, either from wt or H2B-GFP mice, immunostained for CD63 and GFP. Scale bars: 0.1 µm. Figure S6 in Supplementary Material shows electron micrographs of extracellular vesicles immunostained for histone H2A and H3.

    Journal: Frontiers in Immunology

    Article Title: LPS-Challenged Macrophages Release Microvesicles Coated With Histones

    doi: 10.3389/fimmu.2018.01463

    Figure Lengend Snippet: Histones are present on the outer surface of microvesicles and exosomes (MEs). (A) H2B-GFP mice were exposed to doxycycline to induce H2B-GFP expression. Three weeks later, the mice were sacrificed and their bone marrows were harvested and cultured without doxycycline. In this way, bone marrow-derived macrophages (BMDMs) only contained H2B-GFP synthesized several weeks earlier, and all H2B-GFP was chromatin-associated. H2B-GFP BMDMs were then challenged with LPS, and imaged by stochastic optical reconstruction microscopy. H2B-GFP is green; cell membranes were stained red with concanavalin A-TRITC. Zooms of the regions inside the white squares are shown. (B) Western blot of macrobodies and MEs treated with trypsin in the presence or not of detergent (Triton X-100). The electron micrographs show representative MEs before and after trypsinization. (C) Transmission electron microscopy images of exosomes from LPS-challenged BMDMs, either from wt or H2B-GFP mice, immunostained for CD63 and GFP. Scale bars: 0.1 µm. Figure S6 in Supplementary Material shows electron micrographs of extracellular vesicles immunostained for histone H2A and H3.

    Article Snippet: Blocked membranes were probed with the following antibodies in TBS-T at 4°C for 16–24 h: rabbit Chip-Grade anti-H3 antibody (1:1,000; ab1791, Abcam plc, Cambridge, UK), anti-H2A (1:1,000; ab18255, Abcam plc, Cambridge, UK), anti-H2B (1:250; Upstate #07-371, Merck Millipore, KGaA, Darmstadt, Germany), mouse anti-beta-actin (1:2,000; A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-flotillin (1:250; #610820, BD Bioscience, San Jose, CA, USA), anti-TSG101 (1:1,000; ab30871, Abcam plc, Cambridge, UK), anti-citrullinated H3 (1:1,000; ab5103, Abcam plc, Cambridge, UK), anti-HLA IA/IE-PE (BD PharmingenTM).

    Techniques: Mouse Assay, Expressing, Cell Culture, Derivative Assay, Synthesized, Microscopy, Staining, Western Blot, Transmission Assay, Electron Microscopy

    LPS-challenged bone marrow-derived macrophages (BMDMs) lose histones from the nucleus and modify their chromatin architecture. (A) Histone H3 quantification in BMDMs at 0, 1, 2, and 4 h after LPS challenge. (B) Histone H3, H2A, and H2B quantification in untreated and LPS-treated BMDMs. The bar plots in (A,B) show the fluorimetric quantification of the bands that were normalized to beta-actin, and by setting to “1” the histone amount in BMDMs not challenged with LPS. Error bars represent SDs. One-way ANOVA with Dunnett’s posttest; * p

    Journal: Frontiers in Immunology

    Article Title: LPS-Challenged Macrophages Release Microvesicles Coated With Histones

    doi: 10.3389/fimmu.2018.01463

    Figure Lengend Snippet: LPS-challenged bone marrow-derived macrophages (BMDMs) lose histones from the nucleus and modify their chromatin architecture. (A) Histone H3 quantification in BMDMs at 0, 1, 2, and 4 h after LPS challenge. (B) Histone H3, H2A, and H2B quantification in untreated and LPS-treated BMDMs. The bar plots in (A,B) show the fluorimetric quantification of the bands that were normalized to beta-actin, and by setting to “1” the histone amount in BMDMs not challenged with LPS. Error bars represent SDs. One-way ANOVA with Dunnett’s posttest; * p

    Article Snippet: Blocked membranes were probed with the following antibodies in TBS-T at 4°C for 16–24 h: rabbit Chip-Grade anti-H3 antibody (1:1,000; ab1791, Abcam plc, Cambridge, UK), anti-H2A (1:1,000; ab18255, Abcam plc, Cambridge, UK), anti-H2B (1:250; Upstate #07-371, Merck Millipore, KGaA, Darmstadt, Germany), mouse anti-beta-actin (1:2,000; A5441, Sigma-Aldrich, St. Louis, MO, USA), anti-flotillin (1:250; #610820, BD Bioscience, San Jose, CA, USA), anti-TSG101 (1:1,000; ab30871, Abcam plc, Cambridge, UK), anti-citrullinated H3 (1:1,000; ab5103, Abcam plc, Cambridge, UK), anti-HLA IA/IE-PE (BD PharmingenTM).

    Techniques: Derivative Assay

    Impairment of Aurora-B-mediated activities on H2B and HIPK2 induces cytokinesis failure. a , b HeLa cells were transfected with vectors carrying the indicated GFP-HIPK2 forms and stained 24 h post-transfection with anti-p-Histone H2B-S14 and Hoechst. The percentages of midbodies positive for H2B-S14 P staining in the transfected populations a and the percentages of binucleated cells also in the transfected populations b are reported as mean ± SD of two different experiments. c HF were transfected with vectors carrying GFP-tagged H2B-WT or the non-phosphorylatable H2B-S32A derivative. Cells were stained with anti-β-tubulin Ab and Hoechst. The percentages of binucleated cells in the GFP-positive populations were evaluated at the indicated time-points post-transfection and reported as fold change relative to that of GFP-H2B-WT in two different experiments. * p

    Journal: Oncogene

    Article Title: HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

    doi: 10.1038/s41388-018-0191-6

    Figure Lengend Snippet: Impairment of Aurora-B-mediated activities on H2B and HIPK2 induces cytokinesis failure. a , b HeLa cells were transfected with vectors carrying the indicated GFP-HIPK2 forms and stained 24 h post-transfection with anti-p-Histone H2B-S14 and Hoechst. The percentages of midbodies positive for H2B-S14 P staining in the transfected populations a and the percentages of binucleated cells also in the transfected populations b are reported as mean ± SD of two different experiments. c HF were transfected with vectors carrying GFP-tagged H2B-WT or the non-phosphorylatable H2B-S32A derivative. Cells were stained with anti-β-tubulin Ab and Hoechst. The percentages of binucleated cells in the GFP-positive populations were evaluated at the indicated time-points post-transfection and reported as fold change relative to that of GFP-H2B-WT in two different experiments. * p

    Article Snippet: The following Abs were employed: anti-TSG101, anti-cep55, anti-MgcRacGAP1, anti-p-histone-H2B-S32, and anti-Aurora-B (Abcam); anti-PRC1, anti-Alix, anti-PCNA, and anti-GST (Santa Cruz); anti-GFP (Roche); anti-p-histone-H2B-S14 (Cell Signaling).

    Techniques: Transfection, Staining

    Aurora-B phosphorylates H2B at Ser32. a Cold in vitro kinase assays were performed and H2B-S32 P detected using anti-p-Histone H2B-Ser32 Ab. Ponceau staining and immunoblot with anti-H2B Ab were used as loading controls. n = three independent experiment. b Unsynchronized HeLa cells were treated with Hesperadin or DMSO for 80 min and stained with anti-p-Histone H2B-S32 and anti-β-tubulin Abs. Hoechst was used to stain DNA (blue). Representative images of cells at telophase stage are reported. Scale bar is 5 μm. c Representative confocal images of HeLa cells at indicated stages of mitosis and cytokinesis showing colocalization between H2B-S32 P (green) and Aurora-B (red) n = four independent experiment. DNA was stained with Red-Dot2 far-red (pseudo-colored blue); scale bar is 10 μm. d , e HeLa cells stably expressing GFP-H2B WT or its derivative GFP-H2B-S32A at passage two after the establishment of stable populations (i.e., 15 days after blastacidin treatment) were enriched in telophase and protein lysates were obtained from TCEs (T-TCE) or after midbody isolation (MI). Untrasfected HeLa cells were used as negative control. The indicated proteins were analyzed by WB. Immunoblots with anti-PRC1 and anti-p-Histone H2B-S14 Abs were used as positive control to verify the quality of midbody isolation. Immunoblot with anti-PCNA Ab was used as control to evaluate nuclear contamination. Representative WBs are shown. In d , samples were loaded on the same gel and processed on the same filter. Blot was vertically cropped to eliminate non-related samples

    Journal: Oncogene

    Article Title: HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

    doi: 10.1038/s41388-018-0191-6

    Figure Lengend Snippet: Aurora-B phosphorylates H2B at Ser32. a Cold in vitro kinase assays were performed and H2B-S32 P detected using anti-p-Histone H2B-Ser32 Ab. Ponceau staining and immunoblot with anti-H2B Ab were used as loading controls. n = three independent experiment. b Unsynchronized HeLa cells were treated with Hesperadin or DMSO for 80 min and stained with anti-p-Histone H2B-S32 and anti-β-tubulin Abs. Hoechst was used to stain DNA (blue). Representative images of cells at telophase stage are reported. Scale bar is 5 μm. c Representative confocal images of HeLa cells at indicated stages of mitosis and cytokinesis showing colocalization between H2B-S32 P (green) and Aurora-B (red) n = four independent experiment. DNA was stained with Red-Dot2 far-red (pseudo-colored blue); scale bar is 10 μm. d , e HeLa cells stably expressing GFP-H2B WT or its derivative GFP-H2B-S32A at passage two after the establishment of stable populations (i.e., 15 days after blastacidin treatment) were enriched in telophase and protein lysates were obtained from TCEs (T-TCE) or after midbody isolation (MI). Untrasfected HeLa cells were used as negative control. The indicated proteins were analyzed by WB. Immunoblots with anti-PRC1 and anti-p-Histone H2B-S14 Abs were used as positive control to verify the quality of midbody isolation. Immunoblot with anti-PCNA Ab was used as control to evaluate nuclear contamination. Representative WBs are shown. In d , samples were loaded on the same gel and processed on the same filter. Blot was vertically cropped to eliminate non-related samples

    Article Snippet: The following Abs were employed: anti-TSG101, anti-cep55, anti-MgcRacGAP1, anti-p-histone-H2B-S32, and anti-Aurora-B (Abcam); anti-PRC1, anti-Alix, anti-PCNA, and anti-GST (Santa Cruz); anti-GFP (Roche); anti-p-histone-H2B-S14 (Cell Signaling).

    Techniques: In Vitro, Staining, Stable Transfection, Expressing, Isolation, Negative Control, Western Blot, Positive Control