normal mouse igg Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore normal mouse igg
    TGF-β1 induces histone <t>H3K9/14Ac</t> at the PAI-1 and p21 promoters. A : upstream promoter regions of the PAI-I, p21, and CypA genes. Location of primers used for the chromatin immunoprecipitation (ChIP)-qPCR assays labeled P1, P2, or P3 are indicated by short arrows. RMCs were treated with TGF-β1 (5 ng/ml) for indicated time periods up to 6 h. The control samples were harvested at 6 h along with samples treated with TGF-β1 for 6 h. B – D : ChIP assays were performed with H3K9/14Ac antibody, and ChIP-enriched DNA was amplified with primers spanning the 3 regions of the PAI-1 promoter. E : ChIP analysis of H3K9/14Ac on the p21 promoter. F : ChIP analysis of H3K9/14Ac on the CypA promoter region. <t>IgG</t> was used as the antibody control. The bar graphs represent relative enrichment levels normalized to the input and expressed as fold over the untreated controls (means ± SE, n = 3). P, ChIP primers; TSS, transcription start site; SBE, Smad Binding Element; Sp1, Sp1 binding element. * P
    Normal Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Millipore
    Average 99 stars, based on 1703 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    95
    Abcam normal mouse igg
    CXCL8 promoted the migration and invasion of the TE-8 cells (A) (i) For the transwell migration assay, TE-8 cells were plated on the transwell in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. rhCXCL8 was added in the upper chamber at 10 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 24 h. The migrated cells on the underside of the membrane were stained by Diff-Quik and counted. The results are mean ± SEM. Scale bar, 100 μm. (ii) For the transwell invasion assay, TE-8 cells were seeded on a transwell coated with matrigel in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. Recombinant human CXCL8 was added in the transwell at 100 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 48 h. The invaded cells on the underside of the membrane were stained by Diff-Quik and counted. (B) (i) TE-8 cells were plated on the upper chamber with or without rhCXCL8 at 10 ng/ml combined with the inhibitor against PI3K (LY294002, 10 μM) or MEK1/2 (PD98059, 10 μM). DMSO (0.2 μl/ml) was added to negative control. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell combined with LY294002 (10 μM) or PD98059 (10 μM). DMSO (0.2 μl/ml) was added to negative control. After 48 h, the invaded cells were counted. (C) TE-8 cells were transfected with 20 nM siRNA targeting <t>CXCR1</t> and CXCR2 . siNC was transfected to TE-8 as negative control. Effective knockdown of CXCR1 and CXCR2 was confirmed by western blotting using antibodies against CXCR1 and CXCR2. (D) (i) CXCR1 - or CXCR2- silenced TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml. After 24 h, the migrated cells were counted. (ii) CXCR1 - or CXCR2 -silenced TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell at 100 ng/ml. After 48 h, the invaded cells were counted. (E) (i) TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse <t>IgG</t> was added to negative control. The concentrations of neutralizing antibodies were based on the manufacturer's instructions. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the upper transwell at 100 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse IgG was added to negative control. After 48 h, the invaded cells were counted. NS, not significant; * p
    Normal Mouse Igg, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Abcam
    Average 95 stars, based on 499 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    95/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology normal mouse igg
    MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against <t>CREB,</t> RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and <t>IgG</t> (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.
    Normal Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 3654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Santa Cruz Biotechnology
    Average 93 stars, based on 3654 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    93
    Agilent technologies normal mouse igg
    Toxicity of CSF samples from sALS toward motor neuronal cell line comes from misfolded SOD1. ( a ) Differentiated NSC-34 cells were exposed to (white bars) the CSF samples, (gray bars) the CSF samples preabsorbed with normal mouse <t>IgG,</t> and (black bars) the CSF samples preabsorbed with mouse monoclonal <t>C4F6</t> antibody for 48 h, and the cell viability was assayed with Cell Counting Kit-8. Each of the CSF samples was tested in duplicate, and the data are shown as the averaged cell viability relative to that of the negative control, in which PBS instead of CSF samples was exposed to the cells. ( b - g ) Representative images of the differentiated NSC-34 cells exposed to the CSF samples from ( b - d ) non-ND (C-15) and ( e - g ) sALS (ALS13) are shown. The cells were incubated with ( b , e ) the CSF samples, ( c , f ) the CSF samples preabsorbed with mouse IgG, and ( d , g ) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody. The dying cells were stained with DAPI (shown in blue)
    Normal Mouse Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Agilent technologies
    Average 93 stars, based on 84 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    93
    The Jackson Laboratory normal mouse igg
    Toxicity of CSF samples from sALS toward motor neuronal cell line comes from misfolded SOD1. ( a ) Differentiated NSC-34 cells were exposed to (white bars) the CSF samples, (gray bars) the CSF samples preabsorbed with normal mouse <t>IgG,</t> and (black bars) the CSF samples preabsorbed with mouse monoclonal <t>C4F6</t> antibody for 48 h, and the cell viability was assayed with Cell Counting Kit-8. Each of the CSF samples was tested in duplicate, and the data are shown as the averaged cell viability relative to that of the negative control, in which PBS instead of CSF samples was exposed to the cells. ( b - g ) Representative images of the differentiated NSC-34 cells exposed to the CSF samples from ( b - d ) non-ND (C-15) and ( e - g ) sALS (ALS13) are shown. The cells were incubated with ( b , e ) the CSF samples, ( c , f ) the CSF samples preabsorbed with mouse IgG, and ( d , g ) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody. The dying cells were stained with DAPI (shown in blue)
    Normal Mouse Igg, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/The Jackson Laboratory
    Average 93 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    93
    Jackson Immuno normal mouse igg
    Transcription factor binding sites in <t>H4K20ac-enriched</t> sequences. ( a ) The top 20 binding motifs that were observed in H4K20ac-enriched sequences (upper). University of California Santa Cruz’s Transcription Factor Binding Sites conserved track (hg19, tfbsConsSites) was used for the location definition of cis-elements on the human genome. H4K20ac enrichment across factor binding motif sites for AP2-α (middle) and NRSF/REST (lower) are shown. See Supplementary Fig. S4 for other factors. ( b ) H4K20ac distribution across transcription factor binding sites in HeLa cells by ChIP-seq (AP2-alpha and NRSF). See Supplementary Fig. S4 for other factors. ( c ) ChIP-western. Crosslinked HeLa cell chromatin was immunoprecipitated with control mouse <t>IgG,</t> anti-H4K20ac, and anti-H3K9ac. After de-crosslinking, the input (1×, 1/10×, and 1/100×) and the immunoprecipitates were separated in SDS-polyacrylamide gels and probed with the indicated antibodies. NRSF/REST signal was observed in H4K20ac ChIPed sample. The immunoblotting pictures were cropped from original full length immunoblots ( Supplementary Fig. S5 ). The gels were run under the same experimental conditions. ( d ) Schematic drawing of a possible H4K20ac function in transcriptional repression. Binding of transcriptional activators may be inhibited by H4K20ac.
    Normal Mouse Igg, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 93/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Jackson Immuno
    Average 93 stars, based on 217 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    91
    Merck & Co normal mouse igg
    TGFB2-AS1 Interacts with EED (A) Predicted secondary structure of TGFB2-AS1 generated by RNAfold. The 5′ and 3′ ends and break-points (arrowheads) of fragments used in RNA pull-down are marked with nucleotide numbers: 1–200, 201–395, and 396–557. Each nucleotide is color-coded (see color scale): dark blue (probability 0) indicates the lowest and dark red (probability 1) the highest base-pairing probability; for unpaired regions, the color highlights the probability of being unpaired. (B) Schematic illustration of the RNA pull-down assay followed by mass spectrometry analysis. (C) In vitro transcribed biotinylated F-luciferase ( F-luc ) mRNA and TGFB2-AS1 RNA analyzed by agarose gel electrophoresis and molecular size markers (nt). (D) Total protein numbers and examples of proteins interacting with TGFB2-AS1 or F-luc mRNA, identified by mass spectrometry in nuclear lysates of HaCaT cells, stimulated with or without TGF-β for 1 h. EED is highlighted (red) and Smad3 is in brackets because of low statistical coverage. (E) RNA pull-down assay using biotinylated full-length (FL) or different TGFB2-AS1 fragments immobilized on streptavidin beads and lysates from HA-EED overexpressing HEK293T cells. The same cell lysate was applied to each specific RNA. Biotinylated TGFB2-AS1 RNA fragments were analyzed by agarose gel electrophoresis (top). The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibody. Representative immunoblots out of two independent experiments with molecular size markers (kDa). (F) In vitro transcribed biotinylated anti-TGFB2-AS1 and TGFB2-AS1 RNAs were analyzed using agarose gel electrophoresis along with molecular size markers (nt). (G) Quantitative real-time PCR to determine mRNA levels of EED in HEK293T cells transiently transfected with siEED or siControl. The error bars represent SD from three different experiments. (H) RNA pull-down assay using biotinylated (biot-) anti-TGFB2-AS1 and TGFB2-AS1 RNAs (black background) immobilized on streptavidin beads and lysates from HEK293T cells transiently transfected with control (−) or specific siRNA targeting EED (siEED; +) and overexpressing HA-EED, HA-EZH2, and <t>HA-SUZ12.</t> The same cell lysate was applied to each specific RNA. The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibodies. Representative immunoblots out of three independent experiments, with two different exposures (exp.) for HA-EED and molecular size markers (kDa). A star indicates a non-specific protein band. Dotted lines serve orientation and alignment (the immunoblots were not disrupted). (I) RIP of HaCaT lysates using antibodies against endogenous EED, EZH2, SUZ12, or control <t>IgG.</t> Error bars represent SD from three different experiments. Corresponding immunoblots indicate the immunoprecipitated IgG or specific protein (marked by arrowheads) and molecular size markers (kDa). (J) RIP of HaCaT lysates after transient transfection with siEED or siControl, using antibody against endogenous EZH2 or control IgG. Error bars represent SD from three different experiments. (K) Immunoprecipitation of endogenous SUZ12 (or control IgG) in HEK293T cells transiently transfected with the indicated expression constructs and stimulated or not with TGF-β1 for 1 h, followed by immunoblotting for endogenous SUZ12, endogenous EZH2, or transfected HA-EED. Immunoblots of corresponding total cell lysates (TCLs) for the same three proteins in addition to p-Smad2 (indicator of TGF-β stimulation), total Smad2/3, and loading control β-actin, along with molecular size markers (kDa). Stars indicate non-specific protein bands. Representative immunoblots out of three independent experiments are shown.
    Normal Mouse Igg, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Merck & Co
    Average 91 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    92
    Millipore nc normal mouse igg
    TGFB2-AS1 Interacts with EED (A) Predicted secondary structure of TGFB2-AS1 generated by RNAfold. The 5′ and 3′ ends and break-points (arrowheads) of fragments used in RNA pull-down are marked with nucleotide numbers: 1–200, 201–395, and 396–557. Each nucleotide is color-coded (see color scale): dark blue (probability 0) indicates the lowest and dark red (probability 1) the highest base-pairing probability; for unpaired regions, the color highlights the probability of being unpaired. (B) Schematic illustration of the RNA pull-down assay followed by mass spectrometry analysis. (C) In vitro transcribed biotinylated F-luciferase ( F-luc ) mRNA and TGFB2-AS1 RNA analyzed by agarose gel electrophoresis and molecular size markers (nt). (D) Total protein numbers and examples of proteins interacting with TGFB2-AS1 or F-luc mRNA, identified by mass spectrometry in nuclear lysates of HaCaT cells, stimulated with or without TGF-β for 1 h. EED is highlighted (red) and Smad3 is in brackets because of low statistical coverage. (E) RNA pull-down assay using biotinylated full-length (FL) or different TGFB2-AS1 fragments immobilized on streptavidin beads and lysates from HA-EED overexpressing HEK293T cells. The same cell lysate was applied to each specific RNA. Biotinylated TGFB2-AS1 RNA fragments were analyzed by agarose gel electrophoresis (top). The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibody. Representative immunoblots out of two independent experiments with molecular size markers (kDa). (F) In vitro transcribed biotinylated anti-TGFB2-AS1 and TGFB2-AS1 RNAs were analyzed using agarose gel electrophoresis along with molecular size markers (nt). (G) Quantitative real-time PCR to determine mRNA levels of EED in HEK293T cells transiently transfected with siEED or siControl. The error bars represent SD from three different experiments. (H) RNA pull-down assay using biotinylated (biot-) anti-TGFB2-AS1 and TGFB2-AS1 RNAs (black background) immobilized on streptavidin beads and lysates from HEK293T cells transiently transfected with control (−) or specific siRNA targeting EED (siEED; +) and overexpressing HA-EED, HA-EZH2, and <t>HA-SUZ12.</t> The same cell lysate was applied to each specific RNA. The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibodies. Representative immunoblots out of three independent experiments, with two different exposures (exp.) for HA-EED and molecular size markers (kDa). A star indicates a non-specific protein band. Dotted lines serve orientation and alignment (the immunoblots were not disrupted). (I) RIP of HaCaT lysates using antibodies against endogenous EED, EZH2, SUZ12, or control <t>IgG.</t> Error bars represent SD from three different experiments. Corresponding immunoblots indicate the immunoprecipitated IgG or specific protein (marked by arrowheads) and molecular size markers (kDa). (J) RIP of HaCaT lysates after transient transfection with siEED or siControl, using antibody against endogenous EZH2 or control IgG. Error bars represent SD from three different experiments. (K) Immunoprecipitation of endogenous SUZ12 (or control IgG) in HEK293T cells transiently transfected with the indicated expression constructs and stimulated or not with TGF-β1 for 1 h, followed by immunoblotting for endogenous SUZ12, endogenous EZH2, or transfected HA-EED. Immunoblots of corresponding total cell lysates (TCLs) for the same three proteins in addition to p-Smad2 (indicator of TGF-β stimulation), total Smad2/3, and loading control β-actin, along with molecular size markers (kDa). Stars indicate non-specific protein bands. Representative immunoblots out of three independent experiments are shown.
    Nc Normal Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nc normal mouse igg/product/Millipore
    Average 92 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    nc normal mouse igg - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    99
    Bio-Rad normal mouse igg
    <t>Matrigel</t> invasion assay: polyclonal anti-CagA antibodies, but not normal mouse <t>IgG</t> impair trophoblast cell invasiveness in a dose-dependent manner.
    Normal Mouse Igg, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Bio-Rad
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    88
    Santa Cruz Biotechnology normal mouse igg antibodies
    In vivo interaction of <t>p53</t> with the proximal region of the SALL2 P2 promoter. A . Relative positions of the PCR primers for amplification of the proximal and distal regions of the SALL2 P2 promoter and the intron region are shown schematically, the arrows represent the primer set positions refer to the ATG of Exon 1A. The circles and numbers indicate the location of the p53 half sites described in Figure 1A . B . ChIP analysis for the presence of p53 on the proximal and distal regions of the SALL2 P2 promoter and the intron region after 24h treatment with doxorubicin, the arrows show the expected band size. C . ChIP analysis for the presence of p53 on the proximal region of Sall2 promoter after 12 h treatment with doxorubicin. The presence of p53 on the p21 promoter was used as positive control, and purified mouse <t>IgG</t> was used as control antibody. D . Densitometry analysis of a representative ChIP experiment on the p21 promoter and the proximal region of SALL2 promoter after 12 h treatment. Values are expressed as percent of input. E . ChIP analysis for the presence of p53 on the proximal region after 24h treatment with doxorubicin. G . Densitometry analysis of a representative ChIP experiment on the proximal region of SALL2 promoter after 24h with doxorubicin. All experiments were performed in triplicate.
    Normal Mouse Igg Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg antibodies/product/Santa Cruz Biotechnology
    Average 88 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg antibodies - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    93
    Thermo Fisher normal mouse igg
    IL-4 and STAT6 inhibit <t>PPARα</t> transcriptional activity. ( A ) Suppression of PPARα transcriptional activity by STAT6 and IL-4. CV-1 cells were transfected with reporter plasmid (PPRE 3 -tk-Luc, 100 ng) and expression plasmids for PPARα (25 ng) and STAT6 (varying amounts). ( B ) Coimmunoprecipitation of STAT6 and PPARα. Liver lysates from treated animals were immunoprecipitated with anti-PPARα antibody and immunoblotted for STAT6. ( C ) ChIP analysis of PPARα target genes. Chromatin fragments were precipitated from hepatocytes treated with vehicle or Wy14643 (Wy) in the presence or absence of IL-4. Regions flanking the PPARα binding sites on Cyp4a10 and Acot1 promoters were amplified by qPCR and data were normalized to <t>IgG</t> control. ( D and E ) Activation of STAT6 by IL-4 represses PPARα transcriptional activity. Quantitative RT-PCR analyses of PPARα target genes in primary hepatocytes. Error bars are displayed as mean ± SEM ( n = 3–4 for each mouse group). * P
    Normal Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/Thermo Fisher
    Average 93 stars, based on 334 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

    91
    Santa Cruz Biotechnology isotype normal mouse igg
    IL-4 and STAT6 inhibit <t>PPARα</t> transcriptional activity. ( A ) Suppression of PPARα transcriptional activity by STAT6 and IL-4. CV-1 cells were transfected with reporter plasmid (PPRE 3 -tk-Luc, 100 ng) and expression plasmids for PPARα (25 ng) and STAT6 (varying amounts). ( B ) Coimmunoprecipitation of STAT6 and PPARα. Liver lysates from treated animals were immunoprecipitated with anti-PPARα antibody and immunoblotted for STAT6. ( C ) ChIP analysis of PPARα target genes. Chromatin fragments were precipitated from hepatocytes treated with vehicle or Wy14643 (Wy) in the presence or absence of IL-4. Regions flanking the PPARα binding sites on Cyp4a10 and Acot1 promoters were amplified by qPCR and data were normalized to <t>IgG</t> control. ( D and E ) Activation of STAT6 by IL-4 represses PPARα transcriptional activity. Quantitative RT-PCR analyses of PPARα target genes in primary hepatocytes. Error bars are displayed as mean ± SEM ( n = 3–4 for each mouse group). * P
    Isotype Normal Mouse Igg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isotype normal mouse igg/product/Santa Cruz Biotechnology
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    isotype normal mouse igg - by Bioz Stars, 2020-07
    91/100 stars
      Buy from Supplier

    90
    R&D Systems normal mouse igg
    <t>TGF-β1</t> and TGF-β2, but not other pro-fibrotic cytokines, exclusively induces the expression of EMT markers in Müller glial cells. ( A–D ) Müller glial cells were treated with TGF-β1, TGF-β2, BMP-4, CTGF, GDNF, NGF, FGF2, and PDGF-BB at the dose of 10 ng/ml for 24 hours, and ACTA2 ( A ), TAGLN ( B ), COL1A1 ( C ), and FN1 ( D ) expression levels were analyzed. E-H , Müller glial cells were treated for 24 hours with the reaction mixture of 10 ng/ml TGF-β1 preincubated with anti-TGF-β1 neutralizing antibody or control normal <t>IgG</t> at the dose of 200 ng/ml for 15 minutes, and ACTA2 ( E ), TAGLN ( F ), COL1A1 ( G ), and FN1 ( H ) gene expression levels were analyzed. n = 5–6 per group, * p
    Normal Mouse Igg, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/normal mouse igg/product/R&D Systems
    Average 90 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    normal mouse igg - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    Image Search Results


    TGF-β1 induces histone H3K9/14Ac at the PAI-1 and p21 promoters. A : upstream promoter regions of the PAI-I, p21, and CypA genes. Location of primers used for the chromatin immunoprecipitation (ChIP)-qPCR assays labeled P1, P2, or P3 are indicated by short arrows. RMCs were treated with TGF-β1 (5 ng/ml) for indicated time periods up to 6 h. The control samples were harvested at 6 h along with samples treated with TGF-β1 for 6 h. B – D : ChIP assays were performed with H3K9/14Ac antibody, and ChIP-enriched DNA was amplified with primers spanning the 3 regions of the PAI-1 promoter. E : ChIP analysis of H3K9/14Ac on the p21 promoter. F : ChIP analysis of H3K9/14Ac on the CypA promoter region. IgG was used as the antibody control. The bar graphs represent relative enrichment levels normalized to the input and expressed as fold over the untreated controls (means ± SE, n = 3). P, ChIP primers; TSS, transcription start site; SBE, Smad Binding Element; Sp1, Sp1 binding element. * P

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Involvement of p300/CBP and epigenetic histone acetylation in TGF-?1-mediated gene transcription in mesangial cells

    doi: 10.1152/ajprenal.00523.2012

    Figure Lengend Snippet: TGF-β1 induces histone H3K9/14Ac at the PAI-1 and p21 promoters. A : upstream promoter regions of the PAI-I, p21, and CypA genes. Location of primers used for the chromatin immunoprecipitation (ChIP)-qPCR assays labeled P1, P2, or P3 are indicated by short arrows. RMCs were treated with TGF-β1 (5 ng/ml) for indicated time periods up to 6 h. The control samples were harvested at 6 h along with samples treated with TGF-β1 for 6 h. B – D : ChIP assays were performed with H3K9/14Ac antibody, and ChIP-enriched DNA was amplified with primers spanning the 3 regions of the PAI-1 promoter. E : ChIP analysis of H3K9/14Ac on the p21 promoter. F : ChIP analysis of H3K9/14Ac on the CypA promoter region. IgG was used as the antibody control. The bar graphs represent relative enrichment levels normalized to the input and expressed as fold over the untreated controls (means ± SE, n = 3). P, ChIP primers; TSS, transcription start site; SBE, Smad Binding Element; Sp1, Sp1 binding element. * P

    Article Snippet: Recombinant human TGF-β1 and the pan-specific TGF-β1 antibody (MAB1835) were from R & D Systems (Minneapolis, MN); antibodies against acetylated H3K9/14 (catalog no. 06-599), p300 (05-257), Sp1 (07-645), normal mouse IgG (12-371), and normal rabbit IgG (PP64B) were from Millipore (Billerica, MA); Smad2/3 (8685), acetylated-lysine (9441), HDAC1 (2062), and HDAC5 (2082) antibodies were from Cell Signaling (Danvers, MA); the CBP antibody (ab3652) was from Abcam (Cambridge, MA); the β-actin (A5441) antibody was from Sigma (St. Louis, MO). cDNA kits for reverse transcriptase reactions and SYBR green kits for real-time PCRs were from Applied Biosystems (Foster City, CA).

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

    CXCL8 promoted the migration and invasion of the TE-8 cells (A) (i) For the transwell migration assay, TE-8 cells were plated on the transwell in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. rhCXCL8 was added in the upper chamber at 10 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 24 h. The migrated cells on the underside of the membrane were stained by Diff-Quik and counted. The results are mean ± SEM. Scale bar, 100 μm. (ii) For the transwell invasion assay, TE-8 cells were seeded on a transwell coated with matrigel in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. Recombinant human CXCL8 was added in the transwell at 100 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 48 h. The invaded cells on the underside of the membrane were stained by Diff-Quik and counted. (B) (i) TE-8 cells were plated on the upper chamber with or without rhCXCL8 at 10 ng/ml combined with the inhibitor against PI3K (LY294002, 10 μM) or MEK1/2 (PD98059, 10 μM). DMSO (0.2 μl/ml) was added to negative control. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell combined with LY294002 (10 μM) or PD98059 (10 μM). DMSO (0.2 μl/ml) was added to negative control. After 48 h, the invaded cells were counted. (C) TE-8 cells were transfected with 20 nM siRNA targeting CXCR1 and CXCR2 . siNC was transfected to TE-8 as negative control. Effective knockdown of CXCR1 and CXCR2 was confirmed by western blotting using antibodies against CXCR1 and CXCR2. (D) (i) CXCR1 - or CXCR2- silenced TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml. After 24 h, the migrated cells were counted. (ii) CXCR1 - or CXCR2 -silenced TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell at 100 ng/ml. After 48 h, the invaded cells were counted. (E) (i) TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse IgG was added to negative control. The concentrations of neutralizing antibodies were based on the manufacturer's instructions. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the upper transwell at 100 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse IgG was added to negative control. After 48 h, the invaded cells were counted. NS, not significant; * p

    Journal: Oncotarget

    Article Title: CXCL8 derived from tumor-associated macrophages and esophageal squamous cell carcinomas contributes to tumor progression by promoting migration and invasion of cancer cells

    doi: 10.18632/oncotarget.22526

    Figure Lengend Snippet: CXCL8 promoted the migration and invasion of the TE-8 cells (A) (i) For the transwell migration assay, TE-8 cells were plated on the transwell in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. rhCXCL8 was added in the upper chamber at 10 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 24 h. The migrated cells on the underside of the membrane were stained by Diff-Quik and counted. The results are mean ± SEM. Scale bar, 100 μm. (ii) For the transwell invasion assay, TE-8 cells were seeded on a transwell coated with matrigel in serum-free RPMI-1640 at 5.0 × 10 5 cells/well. Recombinant human CXCL8 was added in the transwell at 100 ng/ml. The cell inserts were set on 24-well plates in RPMI-1640 with 1% FBS for 48 h. The invaded cells on the underside of the membrane were stained by Diff-Quik and counted. (B) (i) TE-8 cells were plated on the upper chamber with or without rhCXCL8 at 10 ng/ml combined with the inhibitor against PI3K (LY294002, 10 μM) or MEK1/2 (PD98059, 10 μM). DMSO (0.2 μl/ml) was added to negative control. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell combined with LY294002 (10 μM) or PD98059 (10 μM). DMSO (0.2 μl/ml) was added to negative control. After 48 h, the invaded cells were counted. (C) TE-8 cells were transfected with 20 nM siRNA targeting CXCR1 and CXCR2 . siNC was transfected to TE-8 as negative control. Effective knockdown of CXCR1 and CXCR2 was confirmed by western blotting using antibodies against CXCR1 and CXCR2. (D) (i) CXCR1 - or CXCR2- silenced TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml. After 24 h, the migrated cells were counted. (ii) CXCR1 - or CXCR2 -silenced TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the transwell at 100 ng/ml. After 48 h, the invaded cells were counted. (E) (i) TE-8 cells were plated on the upper transwell with rhCXCL8 at 10 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse IgG was added to negative control. The concentrations of neutralizing antibodies were based on the manufacturer's instructions. (ii) TE-8 cells were plated on the upper transwell coated with matrigel. rhCXCL8 was added in the upper transwell at 100 ng/ml combined with the neutralizing antibody against CXCR1 (0.2 μg/ml) or CXCR2 (1.0 μg/ml). Mouse IgG was added to negative control. After 48 h, the invaded cells were counted. NS, not significant; * p

    Article Snippet: The neutralizing antibodies were as follows (all from Abcam): normal mouse IgG (#ab188776); mouse antibody against CXCR1 (#ab10400); mouse antibody against CXCR2 (#ab10401); mouse antibody against CXCL8 (#ab18672).

    Techniques: Migration, Transwell Migration Assay, Staining, Diff-Quik, Transwell Invasion Assay, Recombinant, Negative Control, Transfection, Western Blot

    TAM-like PBMo-derived macrophages induced the migration and invasion of TE-8 cells by activating PI3K and MEK1/2 signals through CXCL8 and CXCR1/2 interaction (A) PBMos (1.0 × 10 5 cells/well) were seeded on the lower chamber of 24-well plates with M-CSF (25 ng/ml) for 6 days to induce PBMo-derived macrophages, then incubated with 50% TE-8 CM to induce TAM-like PBMo-derived macrophages. After 2 days, the media were replaced with serum-free media. (i) TE-8 cells were plated on the upper transwell at 5.0 × 10 5 cells/well and set on the plate. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper transwell coated with matrigel at 5.0 × 10 5 cells/well and set on the plate. After 48 h, the invaded cells were counted. Scale bar, 100 μm. (B) (i) TE-8 cells were plated on the upper transwell with the inhibitor against PI3K (LY294002, 10 nM) or MEK1/2 (PD98059, 10 nM). DMSO (0.2 μl/ml) was added to negative control. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper matrigel-coated transwell with LY294002 (10 nM) or PD98059 (10 nM). DMSO (0.2 μl/ml) was added to negative control. After 48 h, the invaded cells were counted. (C) (i) TE-8 cells were plated on the upper transwell with the neutralizing antibodies against CXCR1 (0.2 μg/ml), CXCR2 (1.0 μg/ml) or CXCL8 (1.0 μg/ml). Mouse IgG was added to negative control. After 24 h, the migrated cells were counted. The concentrations of neutralizing antibody were based on manufacturer's instructions. (ii) TE-8 cells were plated on the upper matrigel-coated transwell with the neutralizing antibodies against CXCR1 (0.2 μg/ml), CXCR2 (1.0 μg/ml) or CXCL8 (1.0 μg/ml). Mouse IgG was added to negative control. After 48 h, the invaded cells were counted. The results are mean ± SEM. * p

    Journal: Oncotarget

    Article Title: CXCL8 derived from tumor-associated macrophages and esophageal squamous cell carcinomas contributes to tumor progression by promoting migration and invasion of cancer cells

    doi: 10.18632/oncotarget.22526

    Figure Lengend Snippet: TAM-like PBMo-derived macrophages induced the migration and invasion of TE-8 cells by activating PI3K and MEK1/2 signals through CXCL8 and CXCR1/2 interaction (A) PBMos (1.0 × 10 5 cells/well) were seeded on the lower chamber of 24-well plates with M-CSF (25 ng/ml) for 6 days to induce PBMo-derived macrophages, then incubated with 50% TE-8 CM to induce TAM-like PBMo-derived macrophages. After 2 days, the media were replaced with serum-free media. (i) TE-8 cells were plated on the upper transwell at 5.0 × 10 5 cells/well and set on the plate. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper transwell coated with matrigel at 5.0 × 10 5 cells/well and set on the plate. After 48 h, the invaded cells were counted. Scale bar, 100 μm. (B) (i) TE-8 cells were plated on the upper transwell with the inhibitor against PI3K (LY294002, 10 nM) or MEK1/2 (PD98059, 10 nM). DMSO (0.2 μl/ml) was added to negative control. After 24 h, the migrated cells were counted. (ii) TE-8 cells were plated on the upper matrigel-coated transwell with LY294002 (10 nM) or PD98059 (10 nM). DMSO (0.2 μl/ml) was added to negative control. After 48 h, the invaded cells were counted. (C) (i) TE-8 cells were plated on the upper transwell with the neutralizing antibodies against CXCR1 (0.2 μg/ml), CXCR2 (1.0 μg/ml) or CXCL8 (1.0 μg/ml). Mouse IgG was added to negative control. After 24 h, the migrated cells were counted. The concentrations of neutralizing antibody were based on manufacturer's instructions. (ii) TE-8 cells were plated on the upper matrigel-coated transwell with the neutralizing antibodies against CXCR1 (0.2 μg/ml), CXCR2 (1.0 μg/ml) or CXCL8 (1.0 μg/ml). Mouse IgG was added to negative control. After 48 h, the invaded cells were counted. The results are mean ± SEM. * p

    Article Snippet: The neutralizing antibodies were as follows (all from Abcam): normal mouse IgG (#ab188776); mouse antibody against CXCR1 (#ab10400); mouse antibody against CXCR2 (#ab10401); mouse antibody against CXCL8 (#ab18672).

    Techniques: Derivative Assay, Migration, Incubation, Negative Control

    ChIP assays of calli of wild type on SIM at the WUS locus. A) A diagram of WUS structure, with +1 as the transcription start site, and bars representing the regions examined by ChIP. B) ChIP analysis using antibodies against trimethyl H3K4 and dimethyl H3K9 at 5′ and 3′ regions of WUS in calli of wild type for 20 days on CIM (S0) and 6 days on SIM (S6). C) ChIP analysis using antibodies against H3 acetyl Lys 9 at 5′ and 3′ regions of WUS in calli of wild type (S0, S6). ACTIN was used as a control. The input was chromatin before immunoprecipitation. ‘No AB’ corresponds to chromatin treated with normal mouse IgG as the negative control. Three biological replicates were analyzed and each was tested by three technical replicates, and similar results were obtained. Representative data were shown.

    Journal: PLoS Genetics

    Article Title: DNA Methylation and Histone Modifications Regulate De Novo Shoot Regeneration in Arabidopsis by Modulating WUSCHEL Expression and Auxin Signaling

    doi: 10.1371/journal.pgen.1002243

    Figure Lengend Snippet: ChIP assays of calli of wild type on SIM at the WUS locus. A) A diagram of WUS structure, with +1 as the transcription start site, and bars representing the regions examined by ChIP. B) ChIP analysis using antibodies against trimethyl H3K4 and dimethyl H3K9 at 5′ and 3′ regions of WUS in calli of wild type for 20 days on CIM (S0) and 6 days on SIM (S6). C) ChIP analysis using antibodies against H3 acetyl Lys 9 at 5′ and 3′ regions of WUS in calli of wild type (S0, S6). ACTIN was used as a control. The input was chromatin before immunoprecipitation. ‘No AB’ corresponds to chromatin treated with normal mouse IgG as the negative control. Three biological replicates were analyzed and each was tested by three technical replicates, and similar results were obtained. Representative data were shown.

    Article Snippet: Chromatin samples were immunoprecipitated with antibodies against a negative control normal mouse IgG and H3 dimethyl Lys 9 (both included in EpiQuik™ Plant ChIP Kit), or with antibodies against H3 trimethyl Lys 4 (Abcam USA, Catalogno. ab1012) and H3 acetyl Lys 9 (Abcam USA, Catalogno. ab10812).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

    Knock down of eEF1A1 decreases overall transcription of HSP70. ( A ) Schematic of the HSPA1A locus is shown at the top. Plot shows the occupancy of RNAPII relative to input Ct value in eEF1A1 depleted (Si:eEF1A1) and mock-transfected human fibroblast WI38 cells (Si:NT) as determined by ChIP-QPCR. The values for the IgG control are indicated for each PCR fragment. Media ± SEM values lower than those numbers mean no occupancy. ( B ) Nuclear run-on analysis of HSPA1A and GAPDH transcription in heat shocked (43°C, 20 min) control fibroblasts (NT) or eEF1A1-knocked down cells (eEF1A1) with two unrelated siRNAs (pair A or pair B). Numbers on the left represent the hybridization probe position. Numbers on the right represent % of run-on transcripts in eEF1A1-knocked down cells as compared to mock-transfected cells. ( C ) eEF1A1 controls RNAPII occupancy at the HSP70 promoter after stress as determined by ChIP-QPCR. Panels show the effect of eEF1A1 knock down (si:eEF1A1 [pair B]) on RNAPII occupancy relative to the input Ct value under non-HS conditions (C) or after 30 min of heat shock (HS) on HSP70 and GAPDH promoters. Mock-transfected cells (Si:NT). Data from two independent experiments are presented as the mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.03164.008

    Journal: eLife

    Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

    doi: 10.7554/eLife.03164

    Figure Lengend Snippet: Knock down of eEF1A1 decreases overall transcription of HSP70. ( A ) Schematic of the HSPA1A locus is shown at the top. Plot shows the occupancy of RNAPII relative to input Ct value in eEF1A1 depleted (Si:eEF1A1) and mock-transfected human fibroblast WI38 cells (Si:NT) as determined by ChIP-QPCR. The values for the IgG control are indicated for each PCR fragment. Media ± SEM values lower than those numbers mean no occupancy. ( B ) Nuclear run-on analysis of HSPA1A and GAPDH transcription in heat shocked (43°C, 20 min) control fibroblasts (NT) or eEF1A1-knocked down cells (eEF1A1) with two unrelated siRNAs (pair A or pair B). Numbers on the left represent the hybridization probe position. Numbers on the right represent % of run-on transcripts in eEF1A1-knocked down cells as compared to mock-transfected cells. ( C ) eEF1A1 controls RNAPII occupancy at the HSP70 promoter after stress as determined by ChIP-QPCR. Panels show the effect of eEF1A1 knock down (si:eEF1A1 [pair B]) on RNAPII occupancy relative to the input Ct value under non-HS conditions (C) or after 30 min of heat shock (HS) on HSP70 and GAPDH promoters. Mock-transfected cells (Si:NT). Data from two independent experiments are presented as the mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.03164.008

    Article Snippet: 1 mg of pre-cleared protein was IP overnight at 4°C with 5 μg of anti-eEF1A1 (Millipore, Billerica, MA, USA) or normal rabbit or mouse IgG (Abcam, Cambridge, MA, USA) followed by a 1-hr incubation with 50 μl of Dynabeads protein G (Life Technologies, Van Allen Way Carlsbad, CA, USA).

    Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Hybridization

    DRB decreases RNAPII and eEF1A1 occupancy within the HSP70 gene upon HS. ( A ). Data are the mean ± SEM from three independent experiments. MEF cells expressing eEF1A1 tagged with Cherry and Flag were kept under normal growth conditions (control) or heat-shocked for 40 min at 43°C (HS) or treated with 100 μM DRB for 15 min followed by HS (HS+DRB). ChIP was performed using antibodies for total RNAPII followed by QPCR with primers for the indicated regions. ( B ) DRB decreases eEF1A1 occupancy within the HSP70 gene upon HS. Data are the mean ± SEM from three independent experiments. Transformed MEFs were mock treated and kept under normal growth conditions (control) or heat-shocked for 40 min at 43°C (HS) or treated with 100 μM DRB for 15 min followed by HS (HS+DRB). ChIP was performed using antibodies for eEF1A1 and Flag (in Flag–cells) or IgG followed by QPCR with the indicated primers. DOI: http://dx.doi.org/10.7554/eLife.03164.016

    Journal: eLife

    Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

    doi: 10.7554/eLife.03164

    Figure Lengend Snippet: DRB decreases RNAPII and eEF1A1 occupancy within the HSP70 gene upon HS. ( A ). Data are the mean ± SEM from three independent experiments. MEF cells expressing eEF1A1 tagged with Cherry and Flag were kept under normal growth conditions (control) or heat-shocked for 40 min at 43°C (HS) or treated with 100 μM DRB for 15 min followed by HS (HS+DRB). ChIP was performed using antibodies for total RNAPII followed by QPCR with primers for the indicated regions. ( B ) DRB decreases eEF1A1 occupancy within the HSP70 gene upon HS. Data are the mean ± SEM from three independent experiments. Transformed MEFs were mock treated and kept under normal growth conditions (control) or heat-shocked for 40 min at 43°C (HS) or treated with 100 μM DRB for 15 min followed by HS (HS+DRB). ChIP was performed using antibodies for eEF1A1 and Flag (in Flag–cells) or IgG followed by QPCR with the indicated primers. DOI: http://dx.doi.org/10.7554/eLife.03164.016

    Article Snippet: 1 mg of pre-cleared protein was IP overnight at 4°C with 5 μg of anti-eEF1A1 (Millipore, Billerica, MA, USA) or normal rabbit or mouse IgG (Abcam, Cambridge, MA, USA) followed by a 1-hr incubation with 50 μl of Dynabeads protein G (Life Technologies, Van Allen Way Carlsbad, CA, USA).

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

    eEF1A1 facilitates the export of HSP70 mRNA. ( A and B ) eEF1A1 mediates HSP70 mRNA export upon HS. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. eEF1A1 expression was knocked down by siRNA (pair A for A and pair B for B). At 120 min after HS cells were fixed and HSP70 mRNA detected by FISH. Nucleus stained with DAPI. Merged images show cherry-eEF1A1 in red and nucleus in blue. Gray image shows HSP70 mRNA FISH. Bar = 10 microns. Yellow arrowheads indicate areas with high density of HSP70 mRNA and brighter signal for cherry-eEF1A1. ( C ) Extracts from unstressed ( C ) or heat-shocked (HS) MDA-MB231 cells (60 min at 43°C) were IPed with eEF1A1 antibody or IgG. IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. ( D ) MDA-MB-231 cells were mock-transfected (Si:NT) or knocked-down eEF1A1 (Si:eEF1A1). 48 hr after transfection cells were heat-shocked at 43°C for 30 min and left for recovery at 37°C for 30 min. Cell lysates were laid on 10–55% sucrose gradients and sedimented for 2 hr at 30,000 rpm (43). This allows the separation of larger ribosome-associated mRNAs from translationally inactive sub-polysomal mRNAs. Gradients were collected in 750 μl fractions (the vertical lines in the figure indicate each fraction). Collection was done in an ISCO fraction collector while monitoring the absorbance at 254 nm (y axis). The positions of absorbance peaks corresponding to monosome (40S, 60S, and 80S), and polysomes 1 (2-, 3-, 4-ribosomes per mRNA molecule) and polysomes 2 (5-, 6-, 7-ribosomes per RNA molecule) are indicated. Cells knocked-down for eEF1A1 showed the same polysome profile as Si:NT cells. DOI: http://dx.doi.org/10.7554/eLife.03164.020

    Journal: eLife

    Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

    doi: 10.7554/eLife.03164

    Figure Lengend Snippet: eEF1A1 facilitates the export of HSP70 mRNA. ( A and B ) eEF1A1 mediates HSP70 mRNA export upon HS. MEFs were infected with a lentivirus expressing Cherry-eEF1A1. eEF1A1 expression was knocked down by siRNA (pair A for A and pair B for B). At 120 min after HS cells were fixed and HSP70 mRNA detected by FISH. Nucleus stained with DAPI. Merged images show cherry-eEF1A1 in red and nucleus in blue. Gray image shows HSP70 mRNA FISH. Bar = 10 microns. Yellow arrowheads indicate areas with high density of HSP70 mRNA and brighter signal for cherry-eEF1A1. ( C ) Extracts from unstressed ( C ) or heat-shocked (HS) MDA-MB231 cells (60 min at 43°C) were IPed with eEF1A1 antibody or IgG. IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. ( D ) MDA-MB-231 cells were mock-transfected (Si:NT) or knocked-down eEF1A1 (Si:eEF1A1). 48 hr after transfection cells were heat-shocked at 43°C for 30 min and left for recovery at 37°C for 30 min. Cell lysates were laid on 10–55% sucrose gradients and sedimented for 2 hr at 30,000 rpm (43). This allows the separation of larger ribosome-associated mRNAs from translationally inactive sub-polysomal mRNAs. Gradients were collected in 750 μl fractions (the vertical lines in the figure indicate each fraction). Collection was done in an ISCO fraction collector while monitoring the absorbance at 254 nm (y axis). The positions of absorbance peaks corresponding to monosome (40S, 60S, and 80S), and polysomes 1 (2-, 3-, 4-ribosomes per mRNA molecule) and polysomes 2 (5-, 6-, 7-ribosomes per RNA molecule) are indicated. Cells knocked-down for eEF1A1 showed the same polysome profile as Si:NT cells. DOI: http://dx.doi.org/10.7554/eLife.03164.020

    Article Snippet: 1 mg of pre-cleared protein was IP overnight at 4°C with 5 μg of anti-eEF1A1 (Millipore, Billerica, MA, USA) or normal rabbit or mouse IgG (Abcam, Cambridge, MA, USA) followed by a 1-hr incubation with 50 μl of Dynabeads protein G (Life Technologies, Van Allen Way Carlsbad, CA, USA).

    Techniques: Infection, Expressing, Fluorescence In Situ Hybridization, Staining, Multiple Displacement Amplification, SDS Page, Transfection

    eEF1A1 interacts with HSF1 at HSE upon stress and binds HSP27 promoter. ( A ) Stress-induced formation of the eEF1A1-HSF1 complex in vivo. Extracts from unstressed ( C ) or heat-shocked (HS) MDA-MB231 cells IP with HSF1 antibody or IgG. IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting. ( B ) RNA-dependent formation of the eEF1A-HSF1-HSE complex is specific to heat shock. Top panel shows the super-shift of HSF1-HSE EMSA caused by specified antibodies. MDA-MB-231 cells were heat shocked for 20 min at 43°C (HS) or treated with 80 μM arsenite for 2 hr (right panel). Cell extracts were incubated with HSF1 or eEF1A1 antibodies or IgG (mock). In parallel, cells were also treated with RNAse A prior to EMSA (lower panel). ( C ) Chromatin from MDA-MB231 cells was IP with eEF1A1 and IgG antibodies and amplified by PCR targeting the HSP27 promoter region. Control—cells kept at 37°C, HS—cells heat shocked at 43°C for 30 min. Values below IgG number mean no occupancy. DOI: http://dx.doi.org/10.7554/eLife.03164.011

    Journal: eLife

    Article Title: The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

    doi: 10.7554/eLife.03164

    Figure Lengend Snippet: eEF1A1 interacts with HSF1 at HSE upon stress and binds HSP27 promoter. ( A ) Stress-induced formation of the eEF1A1-HSF1 complex in vivo. Extracts from unstressed ( C ) or heat-shocked (HS) MDA-MB231 cells IP with HSF1 antibody or IgG. IP samples or total protein (Input) were subjected to SDS-PAGE and immunoblotting. ( B ) RNA-dependent formation of the eEF1A-HSF1-HSE complex is specific to heat shock. Top panel shows the super-shift of HSF1-HSE EMSA caused by specified antibodies. MDA-MB-231 cells were heat shocked for 20 min at 43°C (HS) or treated with 80 μM arsenite for 2 hr (right panel). Cell extracts were incubated with HSF1 or eEF1A1 antibodies or IgG (mock). In parallel, cells were also treated with RNAse A prior to EMSA (lower panel). ( C ) Chromatin from MDA-MB231 cells was IP with eEF1A1 and IgG antibodies and amplified by PCR targeting the HSP27 promoter region. Control—cells kept at 37°C, HS—cells heat shocked at 43°C for 30 min. Values below IgG number mean no occupancy. DOI: http://dx.doi.org/10.7554/eLife.03164.011

    Article Snippet: 1 mg of pre-cleared protein was IP overnight at 4°C with 5 μg of anti-eEF1A1 (Millipore, Billerica, MA, USA) or normal rabbit or mouse IgG (Abcam, Cambridge, MA, USA) followed by a 1-hr incubation with 50 μl of Dynabeads protein G (Life Technologies, Van Allen Way Carlsbad, CA, USA).

    Techniques: In Vivo, Multiple Displacement Amplification, SDS Page, Incubation, Amplification, Polymerase Chain Reaction

    MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.

    Journal: Nucleic Acids Research

    Article Title: Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements

    doi: 10.1093/nar/gks1365

    Figure Lengend Snippet: MHCII expression state-related maintenance of the enhanceosome (MCE) and activation-associated histone PTMs in asynchronous or mitotically arrested B lymphoblastoid and epithelial cells. ( A ) Flow cytometric analysis of DNA content upon propidium iodide staining of B lymphoblastoid parental Raji and its CIITA negative derivative RJ2.2.5 cell line (left panels), or epithelial parental HeLa and a CIITA transfectant HeLa (CIITA + ) cell line (right panels), growing asynchronously (upper panels) or mitotically arrested (lower panels) in prometaphase with nocodazole. ( B–E ) Chromatin from the above cell lines was used for ChIP-qPCR analysis with antibodies against CREB, RFX5, NFYA, NFYB, CIITA, TBP, RNA PolII and IgG (B, D) or acetyl-H3, acetyl-H4, H3K4me2, H3K4me3 (C, E) on the DRA promoter. Factor occupancy is expressed as % of input chromatin (B, D) or as relative occupancy of histone H3 or H4 PTMs normalized against total histone H3 or H4 (not shown) respectively (C, E). A value of 1 set for acH3/H3 in asynchronous Raji (C) or HeLa CIITA (E) cells represents 7.5%/0.93% and 11%/4% acH3 and total H3, respectively. Triplicate means and standard deviations are shown.

    Article Snippet: Reagents α-RFX5 was from Rockland, α-NFY-A (H-209) (sc-10779), α-NFYA (G-2) (sc-17753), α-NFY-B (FL-207) (sc-13045), α-CREB (C-21) (sc-186), α-RNA Polymerase II (N-20) (sc-899), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology, α-TBP (ab28175) and α-H3 (ab1791) were from Abcam, α-acetyl H3 (06-599), α-acetyl H4 (06-866), α-H3K4me2 (07-030), α-H3K4me3 (07-473), α-H3S10-Phos (06-570), α-PP2Ac (Cat.# 05-421) and α-CREB (17-600) were from Millipore, α-PP2Ac (#2028) was from Cell Signaling, α-β-tubulin (T4026) was from Sigma-Aldrich, α-GFP was from Minotech, α-JellyRed (α-KillerRed, Cat.# AB962) was from Evrogen, mouse monoclonal α-myc antibody was secreted from the hybridoma cell line Myc-9E10.2 ( ) and anti-CIITA antibody was described earlier ( ).

    Techniques: Expressing, Activation Assay, Flow Cytometry, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The DRA -LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. ( A ) DRA -LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA + ) and HeLa cell lines shown in Figure 2 D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2 B. ( B ) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. ( C ) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.

    Journal: Nucleic Acids Research

    Article Title: Gene-specific factors determine mitotic expression and bookmarking via alternate regulatory elements

    doi: 10.1093/nar/gks1365

    Figure Lengend Snippet: The DRA -LCR/XL4 mediates PP2A recruitment via a direct interaction with NFYA in mitosis. ( A ) DRA -LCR/XL4 factor occupancy using the same chromatin samples, antibodies and primer sets from asynchronous and mitotic HeLa CIITA (CIITA + ) and HeLa cell lines shown in Figure 2 D and E. The inset shows the relative fold change of occupancy of LCR/XL4 over promoter for the indicated factors. Mean and error bars are derived as in Figure 2 B. ( B ) Whole cell extracts of HEK293T cells expressing myc-tagged PP2Ac and the indicated full-length or truncated GFP- or Jelly Red-fusion proteins were used for protein immunoprecipitation experiments using an α-myc antibody. Western blotting was performed using α-JellyRed, α-GFP or α-myc antibodies. Input represents 10% of the lysate used for the immunoprecipitation. ( C ) Whole cell extracts of asynchronous or nocodazole-treated HeLa cells were subjected to immunoprercipitation by α-NFYA and immunoblotted against α-PP2Ac. Shown are the input (5% of immunoprecipitated samples), the normal IgG control and the verification of efficient NFYA immunoprecipitation.

    Article Snippet: Reagents α-RFX5 was from Rockland, α-NFY-A (H-209) (sc-10779), α-NFYA (G-2) (sc-17753), α-NFY-B (FL-207) (sc-13045), α-CREB (C-21) (sc-186), α-RNA Polymerase II (N-20) (sc-899), normal rabbit IgG (sc-2027), normal mouse IgG (sc-2025) were from Santa Cruz Biotechnology, α-TBP (ab28175) and α-H3 (ab1791) were from Abcam, α-acetyl H3 (06-599), α-acetyl H4 (06-866), α-H3K4me2 (07-030), α-H3K4me3 (07-473), α-H3S10-Phos (06-570), α-PP2Ac (Cat.# 05-421) and α-CREB (17-600) were from Millipore, α-PP2Ac (#2028) was from Cell Signaling, α-β-tubulin (T4026) was from Sigma-Aldrich, α-GFP was from Minotech, α-JellyRed (α-KillerRed, Cat.# AB962) was from Evrogen, mouse monoclonal α-myc antibody was secreted from the hybridoma cell line Myc-9E10.2 ( ) and anti-CIITA antibody was described earlier ( ).

    Techniques: Derivative Assay, Expressing, Immunoprecipitation, Western Blot

    Toxicity of CSF samples from sALS toward motor neuronal cell line comes from misfolded SOD1. ( a ) Differentiated NSC-34 cells were exposed to (white bars) the CSF samples, (gray bars) the CSF samples preabsorbed with normal mouse IgG, and (black bars) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody for 48 h, and the cell viability was assayed with Cell Counting Kit-8. Each of the CSF samples was tested in duplicate, and the data are shown as the averaged cell viability relative to that of the negative control, in which PBS instead of CSF samples was exposed to the cells. ( b - g ) Representative images of the differentiated NSC-34 cells exposed to the CSF samples from ( b - d ) non-ND (C-15) and ( e - g ) sALS (ALS13) are shown. The cells were incubated with ( b , e ) the CSF samples, ( c , f ) the CSF samples preabsorbed with mouse IgG, and ( d , g ) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody. The dying cells were stained with DAPI (shown in blue)

    Journal: Molecular Neurodegeneration

    Article Title: Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis

    doi: 10.1186/s13024-019-0341-5

    Figure Lengend Snippet: Toxicity of CSF samples from sALS toward motor neuronal cell line comes from misfolded SOD1. ( a ) Differentiated NSC-34 cells were exposed to (white bars) the CSF samples, (gray bars) the CSF samples preabsorbed with normal mouse IgG, and (black bars) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody for 48 h, and the cell viability was assayed with Cell Counting Kit-8. Each of the CSF samples was tested in duplicate, and the data are shown as the averaged cell viability relative to that of the negative control, in which PBS instead of CSF samples was exposed to the cells. ( b - g ) Representative images of the differentiated NSC-34 cells exposed to the CSF samples from ( b - d ) non-ND (C-15) and ( e - g ) sALS (ALS13) are shown. The cells were incubated with ( b , e ) the CSF samples, ( c , f ) the CSF samples preabsorbed with mouse IgG, and ( d , g ) the CSF samples preabsorbed with mouse monoclonal C4F6 antibody. The dying cells were stained with DAPI (shown in blue)

    Article Snippet: In brief, Dynabeads® M-270 Epox (1 × 108 beads) was first equilibrated in 100 mM sodium phosphate buffer at pH 7.4 and then coupled with either C4F6 (1:50 dilution) or normal mouse IgG (1:50 dilution; Negative control mouse IgG, Dako) in 100 mM sodium phosphate buffer at pH 7.4 containing 1 M ammonium sulfate with slow rotation at 37 °C for 24 h. After the antibody-coated magnetic beads were washed a total of four times with PBS-T, CSF samples (20 or 40 μL) were reacted with the beads with slow rotation at 4 °C for 24 h. The beads were incubated with 100 mM citrate buffer at pH 3.1 to elute misfolded SOD1, and the eluates were analyzed by Western blotting with FL-154 (1:1000 dilution in PBS-T with 5% BSA) followed by the secondary antibody (1:500 dilution in PBS-T; anti-rabbit IgG, HRP-linked antibody, Cell Signaling Technology).

    Techniques: Cell Counting, Negative Control, Incubation, Staining

    Immunoprecipitation experiments confirm the presence of C4F6-reactive SOD1 in CSF of all ALS cases as well as some of PD and PSP cases. The C4F6-crosslinked or mouse IgG-crosslinked magnetic beads were first incubated with CSF (20 μL) at 4 °C for 24 h, and the SOD1 proteins bound to those magnetic beads were then eluted with 10 μL of 100 mM citrate buffer at pH 3.1. The eluates (10 μL) were analyzed by Western blotting with FL-154 antibody. Due to the limited availability of the CSF samples, we could not examine several cases including C-2, C-5, C-6, C-7, C-10, C-16, C-17, ALS1, ALS12, ALS18, ALS19, ALS20, and ALS21

    Journal: Molecular Neurodegeneration

    Article Title: Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis

    doi: 10.1186/s13024-019-0341-5

    Figure Lengend Snippet: Immunoprecipitation experiments confirm the presence of C4F6-reactive SOD1 in CSF of all ALS cases as well as some of PD and PSP cases. The C4F6-crosslinked or mouse IgG-crosslinked magnetic beads were first incubated with CSF (20 μL) at 4 °C for 24 h, and the SOD1 proteins bound to those magnetic beads were then eluted with 10 μL of 100 mM citrate buffer at pH 3.1. The eluates (10 μL) were analyzed by Western blotting with FL-154 antibody. Due to the limited availability of the CSF samples, we could not examine several cases including C-2, C-5, C-6, C-7, C-10, C-16, C-17, ALS1, ALS12, ALS18, ALS19, ALS20, and ALS21

    Article Snippet: In brief, Dynabeads® M-270 Epox (1 × 108 beads) was first equilibrated in 100 mM sodium phosphate buffer at pH 7.4 and then coupled with either C4F6 (1:50 dilution) or normal mouse IgG (1:50 dilution; Negative control mouse IgG, Dako) in 100 mM sodium phosphate buffer at pH 7.4 containing 1 M ammonium sulfate with slow rotation at 37 °C for 24 h. After the antibody-coated magnetic beads were washed a total of four times with PBS-T, CSF samples (20 or 40 μL) were reacted with the beads with slow rotation at 4 °C for 24 h. The beads were incubated with 100 mM citrate buffer at pH 3.1 to elute misfolded SOD1, and the eluates were analyzed by Western blotting with FL-154 (1:1000 dilution in PBS-T with 5% BSA) followed by the secondary antibody (1:500 dilution in PBS-T; anti-rabbit IgG, HRP-linked antibody, Cell Signaling Technology).

    Techniques: Immunoprecipitation, Magnetic Beads, Incubation, Western Blot

    Transcription factor binding sites in H4K20ac-enriched sequences. ( a ) The top 20 binding motifs that were observed in H4K20ac-enriched sequences (upper). University of California Santa Cruz’s Transcription Factor Binding Sites conserved track (hg19, tfbsConsSites) was used for the location definition of cis-elements on the human genome. H4K20ac enrichment across factor binding motif sites for AP2-α (middle) and NRSF/REST (lower) are shown. See Supplementary Fig. S4 for other factors. ( b ) H4K20ac distribution across transcription factor binding sites in HeLa cells by ChIP-seq (AP2-alpha and NRSF). See Supplementary Fig. S4 for other factors. ( c ) ChIP-western. Crosslinked HeLa cell chromatin was immunoprecipitated with control mouse IgG, anti-H4K20ac, and anti-H3K9ac. After de-crosslinking, the input (1×, 1/10×, and 1/100×) and the immunoprecipitates were separated in SDS-polyacrylamide gels and probed with the indicated antibodies. NRSF/REST signal was observed in H4K20ac ChIPed sample. The immunoblotting pictures were cropped from original full length immunoblots ( Supplementary Fig. S5 ). The gels were run under the same experimental conditions. ( d ) Schematic drawing of a possible H4K20ac function in transcriptional repression. Binding of transcriptional activators may be inhibited by H4K20ac.

    Journal: Scientific Reports

    Article Title: Histone H4 lysine 20 acetylation is associated with gene repression in human cells

    doi: 10.1038/srep24318

    Figure Lengend Snippet: Transcription factor binding sites in H4K20ac-enriched sequences. ( a ) The top 20 binding motifs that were observed in H4K20ac-enriched sequences (upper). University of California Santa Cruz’s Transcription Factor Binding Sites conserved track (hg19, tfbsConsSites) was used for the location definition of cis-elements on the human genome. H4K20ac enrichment across factor binding motif sites for AP2-α (middle) and NRSF/REST (lower) are shown. See Supplementary Fig. S4 for other factors. ( b ) H4K20ac distribution across transcription factor binding sites in HeLa cells by ChIP-seq (AP2-alpha and NRSF). See Supplementary Fig. S4 for other factors. ( c ) ChIP-western. Crosslinked HeLa cell chromatin was immunoprecipitated with control mouse IgG, anti-H4K20ac, and anti-H3K9ac. After de-crosslinking, the input (1×, 1/10×, and 1/100×) and the immunoprecipitates were separated in SDS-polyacrylamide gels and probed with the indicated antibodies. NRSF/REST signal was observed in H4K20ac ChIPed sample. The immunoblotting pictures were cropped from original full length immunoblots ( Supplementary Fig. S5 ). The gels were run under the same experimental conditions. ( d ) Schematic drawing of a possible H4K20ac function in transcriptional repression. Binding of transcriptional activators may be inhibited by H4K20ac.

    Article Snippet: For cross-linked ChIP, Dynabeads M-280 sheep anti-mouse IgG (Thermo Fisher Scientific; 500 μl original suspension) were incubated with 10 μg anti-H4K20ac, anti-H3K9ac, or normal mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) in 500 μl RIPA-150 for 3 h at 4 °C with rotation and washed twice with 1 ml RIPA-150.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Western Blot, Immunoprecipitation

    TGFB2-AS1 Interacts with EED (A) Predicted secondary structure of TGFB2-AS1 generated by RNAfold. The 5′ and 3′ ends and break-points (arrowheads) of fragments used in RNA pull-down are marked with nucleotide numbers: 1–200, 201–395, and 396–557. Each nucleotide is color-coded (see color scale): dark blue (probability 0) indicates the lowest and dark red (probability 1) the highest base-pairing probability; for unpaired regions, the color highlights the probability of being unpaired. (B) Schematic illustration of the RNA pull-down assay followed by mass spectrometry analysis. (C) In vitro transcribed biotinylated F-luciferase ( F-luc ) mRNA and TGFB2-AS1 RNA analyzed by agarose gel electrophoresis and molecular size markers (nt). (D) Total protein numbers and examples of proteins interacting with TGFB2-AS1 or F-luc mRNA, identified by mass spectrometry in nuclear lysates of HaCaT cells, stimulated with or without TGF-β for 1 h. EED is highlighted (red) and Smad3 is in brackets because of low statistical coverage. (E) RNA pull-down assay using biotinylated full-length (FL) or different TGFB2-AS1 fragments immobilized on streptavidin beads and lysates from HA-EED overexpressing HEK293T cells. The same cell lysate was applied to each specific RNA. Biotinylated TGFB2-AS1 RNA fragments were analyzed by agarose gel electrophoresis (top). The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibody. Representative immunoblots out of two independent experiments with molecular size markers (kDa). (F) In vitro transcribed biotinylated anti-TGFB2-AS1 and TGFB2-AS1 RNAs were analyzed using agarose gel electrophoresis along with molecular size markers (nt). (G) Quantitative real-time PCR to determine mRNA levels of EED in HEK293T cells transiently transfected with siEED or siControl. The error bars represent SD from three different experiments. (H) RNA pull-down assay using biotinylated (biot-) anti-TGFB2-AS1 and TGFB2-AS1 RNAs (black background) immobilized on streptavidin beads and lysates from HEK293T cells transiently transfected with control (−) or specific siRNA targeting EED (siEED; +) and overexpressing HA-EED, HA-EZH2, and HA-SUZ12. The same cell lysate was applied to each specific RNA. The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibodies. Representative immunoblots out of three independent experiments, with two different exposures (exp.) for HA-EED and molecular size markers (kDa). A star indicates a non-specific protein band. Dotted lines serve orientation and alignment (the immunoblots were not disrupted). (I) RIP of HaCaT lysates using antibodies against endogenous EED, EZH2, SUZ12, or control IgG. Error bars represent SD from three different experiments. Corresponding immunoblots indicate the immunoprecipitated IgG or specific protein (marked by arrowheads) and molecular size markers (kDa). (J) RIP of HaCaT lysates after transient transfection with siEED or siControl, using antibody against endogenous EZH2 or control IgG. Error bars represent SD from three different experiments. (K) Immunoprecipitation of endogenous SUZ12 (or control IgG) in HEK293T cells transiently transfected with the indicated expression constructs and stimulated or not with TGF-β1 for 1 h, followed by immunoblotting for endogenous SUZ12, endogenous EZH2, or transfected HA-EED. Immunoblots of corresponding total cell lysates (TCLs) for the same three proteins in addition to p-Smad2 (indicator of TGF-β stimulation), total Smad2/3, and loading control β-actin, along with molecular size markers (kDa). Stars indicate non-specific protein bands. Representative immunoblots out of three independent experiments are shown.

    Journal: Cell Reports

    Article Title: The TGFB2-AS1 lncRNA Regulates TGF-β Signaling by Modulating Corepressor Activity

    doi: 10.1016/j.celrep.2019.08.028

    Figure Lengend Snippet: TGFB2-AS1 Interacts with EED (A) Predicted secondary structure of TGFB2-AS1 generated by RNAfold. The 5′ and 3′ ends and break-points (arrowheads) of fragments used in RNA pull-down are marked with nucleotide numbers: 1–200, 201–395, and 396–557. Each nucleotide is color-coded (see color scale): dark blue (probability 0) indicates the lowest and dark red (probability 1) the highest base-pairing probability; for unpaired regions, the color highlights the probability of being unpaired. (B) Schematic illustration of the RNA pull-down assay followed by mass spectrometry analysis. (C) In vitro transcribed biotinylated F-luciferase ( F-luc ) mRNA and TGFB2-AS1 RNA analyzed by agarose gel electrophoresis and molecular size markers (nt). (D) Total protein numbers and examples of proteins interacting with TGFB2-AS1 or F-luc mRNA, identified by mass spectrometry in nuclear lysates of HaCaT cells, stimulated with or without TGF-β for 1 h. EED is highlighted (red) and Smad3 is in brackets because of low statistical coverage. (E) RNA pull-down assay using biotinylated full-length (FL) or different TGFB2-AS1 fragments immobilized on streptavidin beads and lysates from HA-EED overexpressing HEK293T cells. The same cell lysate was applied to each specific RNA. Biotinylated TGFB2-AS1 RNA fragments were analyzed by agarose gel electrophoresis (top). The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibody. Representative immunoblots out of two independent experiments with molecular size markers (kDa). (F) In vitro transcribed biotinylated anti-TGFB2-AS1 and TGFB2-AS1 RNAs were analyzed using agarose gel electrophoresis along with molecular size markers (nt). (G) Quantitative real-time PCR to determine mRNA levels of EED in HEK293T cells transiently transfected with siEED or siControl. The error bars represent SD from three different experiments. (H) RNA pull-down assay using biotinylated (biot-) anti-TGFB2-AS1 and TGFB2-AS1 RNAs (black background) immobilized on streptavidin beads and lysates from HEK293T cells transiently transfected with control (−) or specific siRNA targeting EED (siEED; +) and overexpressing HA-EED, HA-EZH2, and HA-SUZ12. The same cell lysate was applied to each specific RNA. The proteins retained on the RNA beads and total cell lysates (TCLs) were analyzed by immunoblotting using the indicated antibodies. Representative immunoblots out of three independent experiments, with two different exposures (exp.) for HA-EED and molecular size markers (kDa). A star indicates a non-specific protein band. Dotted lines serve orientation and alignment (the immunoblots were not disrupted). (I) RIP of HaCaT lysates using antibodies against endogenous EED, EZH2, SUZ12, or control IgG. Error bars represent SD from three different experiments. Corresponding immunoblots indicate the immunoprecipitated IgG or specific protein (marked by arrowheads) and molecular size markers (kDa). (J) RIP of HaCaT lysates after transient transfection with siEED or siControl, using antibody against endogenous EZH2 or control IgG. Error bars represent SD from three different experiments. (K) Immunoprecipitation of endogenous SUZ12 (or control IgG) in HEK293T cells transiently transfected with the indicated expression constructs and stimulated or not with TGF-β1 for 1 h, followed by immunoblotting for endogenous SUZ12, endogenous EZH2, or transfected HA-EED. Immunoblots of corresponding total cell lysates (TCLs) for the same three proteins in addition to p-Smad2 (indicator of TGF-β stimulation), total Smad2/3, and loading control β-actin, along with molecular size markers (kDa). Stars indicate non-specific protein bands. Representative immunoblots out of three independent experiments are shown.

    Article Snippet: The antibodies used for ChIP assays were: anti-H3K27me3 (Abcam, Cambridge, United Kingdom), anti-H3K4me3 (ab8580, Abcam, Cambridge, United Kingdom), anti-EED (Active Motif Europe, La Hulpe, Belgium), anti-EZH2 (Milipore/Merck, Stockholm, Sweden), anti-SUZ12 (Abcam, Cambridge, United Kingdom), anti-Smad2/3 (BD Biosciences-Europe, Stockholm, Sweden) and normal mouse IgG (Millipore/Merck, Stockholm, Sweden).

    Techniques: Generated, Pull Down Assay, Mass Spectrometry, In Vitro, Luciferase, Agarose Gel Electrophoresis, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Immunoprecipitation, Expressing, Construct

    Matrigel invasion assay: polyclonal anti-CagA antibodies, but not normal mouse IgG impair trophoblast cell invasiveness in a dose-dependent manner.

    Journal: Helicobacter

    Article Title: Antibodies Anti-Caga Cross-React with Trophoblast Cells: A Risk Factor for Pre-Eclampsia?

    doi: 10.1111/j.1523-5378.2012.00966.x

    Figure Lengend Snippet: Matrigel invasion assay: polyclonal anti-CagA antibodies, but not normal mouse IgG impair trophoblast cell invasiveness in a dose-dependent manner.

    Article Snippet: To explore whether trophoblast invasiveness was affected by the presence of anti-CagA Ab IgG, we examined the invasion activity using a membrane invasion culture system, in presence of increasing concentrations of anti-Cag Ab. shows the results of a 72 hours in vitro Matrigel invasion assay: polyclonal anti-CagA Ab, but not normal mouse IgG, was able to significantly impair trophoblast cell invasiveness in a dose-dependent manner.

    Techniques: Invasion Assay

    In vivo interaction of p53 with the proximal region of the SALL2 P2 promoter. A . Relative positions of the PCR primers for amplification of the proximal and distal regions of the SALL2 P2 promoter and the intron region are shown schematically, the arrows represent the primer set positions refer to the ATG of Exon 1A. The circles and numbers indicate the location of the p53 half sites described in Figure 1A . B . ChIP analysis for the presence of p53 on the proximal and distal regions of the SALL2 P2 promoter and the intron region after 24h treatment with doxorubicin, the arrows show the expected band size. C . ChIP analysis for the presence of p53 on the proximal region of Sall2 promoter after 12 h treatment with doxorubicin. The presence of p53 on the p21 promoter was used as positive control, and purified mouse IgG was used as control antibody. D . Densitometry analysis of a representative ChIP experiment on the p21 promoter and the proximal region of SALL2 promoter after 12 h treatment. Values are expressed as percent of input. E . ChIP analysis for the presence of p53 on the proximal region after 24h treatment with doxorubicin. G . Densitometry analysis of a representative ChIP experiment on the proximal region of SALL2 promoter after 24h with doxorubicin. All experiments were performed in triplicate.

    Journal: PLoS ONE

    Article Title: Wild Type p53 Transcriptionally Represses the SALL2 Transcription Factor under Genotoxic Stress

    doi: 10.1371/journal.pone.0073817

    Figure Lengend Snippet: In vivo interaction of p53 with the proximal region of the SALL2 P2 promoter. A . Relative positions of the PCR primers for amplification of the proximal and distal regions of the SALL2 P2 promoter and the intron region are shown schematically, the arrows represent the primer set positions refer to the ATG of Exon 1A. The circles and numbers indicate the location of the p53 half sites described in Figure 1A . B . ChIP analysis for the presence of p53 on the proximal and distal regions of the SALL2 P2 promoter and the intron region after 24h treatment with doxorubicin, the arrows show the expected band size. C . ChIP analysis for the presence of p53 on the proximal region of Sall2 promoter after 12 h treatment with doxorubicin. The presence of p53 on the p21 promoter was used as positive control, and purified mouse IgG was used as control antibody. D . Densitometry analysis of a representative ChIP experiment on the p21 promoter and the proximal region of SALL2 promoter after 12 h treatment. Values are expressed as percent of input. E . ChIP analysis for the presence of p53 on the proximal region after 24h treatment with doxorubicin. G . Densitometry analysis of a representative ChIP experiment on the proximal region of SALL2 promoter after 24h with doxorubicin. All experiments were performed in triplicate.

    Article Snippet: Immunoprecipitations were carried out overnight at 4 °C using 3 µg p53 (DO-1, Santa Cruz) or normal mouse IgG antibodies (sc- 2015, Santa Cruz) and 40 µg of chromatin.

    Techniques: In Vivo, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Positive Control, Purification

    IL-4 and STAT6 inhibit PPARα transcriptional activity. ( A ) Suppression of PPARα transcriptional activity by STAT6 and IL-4. CV-1 cells were transfected with reporter plasmid (PPRE 3 -tk-Luc, 100 ng) and expression plasmids for PPARα (25 ng) and STAT6 (varying amounts). ( B ) Coimmunoprecipitation of STAT6 and PPARα. Liver lysates from treated animals were immunoprecipitated with anti-PPARα antibody and immunoblotted for STAT6. ( C ) ChIP analysis of PPARα target genes. Chromatin fragments were precipitated from hepatocytes treated with vehicle or Wy14643 (Wy) in the presence or absence of IL-4. Regions flanking the PPARα binding sites on Cyp4a10 and Acot1 promoters were amplified by qPCR and data were normalized to IgG control. ( D and E ) Activation of STAT6 by IL-4 represses PPARα transcriptional activity. Quantitative RT-PCR analyses of PPARα target genes in primary hepatocytes. Error bars are displayed as mean ± SEM ( n = 3–4 for each mouse group). * P

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

    Article Title: IL-4/STAT6 immune axis regulates peripheral nutrient metabolism and insulin sensitivity

    doi: 10.1073/pnas.1009152108

    Figure Lengend Snippet: IL-4 and STAT6 inhibit PPARα transcriptional activity. ( A ) Suppression of PPARα transcriptional activity by STAT6 and IL-4. CV-1 cells were transfected with reporter plasmid (PPRE 3 -tk-Luc, 100 ng) and expression plasmids for PPARα (25 ng) and STAT6 (varying amounts). ( B ) Coimmunoprecipitation of STAT6 and PPARα. Liver lysates from treated animals were immunoprecipitated with anti-PPARα antibody and immunoblotted for STAT6. ( C ) ChIP analysis of PPARα target genes. Chromatin fragments were precipitated from hepatocytes treated with vehicle or Wy14643 (Wy) in the presence or absence of IL-4. Regions flanking the PPARα binding sites on Cyp4a10 and Acot1 promoters were amplified by qPCR and data were normalized to IgG control. ( D and E ) Activation of STAT6 by IL-4 represses PPARα transcriptional activity. Quantitative RT-PCR analyses of PPARα target genes in primary hepatocytes. Error bars are displayed as mean ± SEM ( n = 3–4 for each mouse group). * P

    Article Snippet: The resultant protein lysate (1.5 mg) was incubated with 4 mg of anti-PPARα monoclonal antibody (ABR) or 4 mg normal mouse IgG (Caltag Laboratories) for 2 h at 4 °C followed by incubation with protein A agrarose beads for 1 h. Immunoprecipitated proteins were analyzed by Western blotting.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay, Amplification, Real-time Polymerase Chain Reaction, Activation Assay, Quantitative RT-PCR

    TGF-β1 and TGF-β2, but not other pro-fibrotic cytokines, exclusively induces the expression of EMT markers in Müller glial cells. ( A–D ) Müller glial cells were treated with TGF-β1, TGF-β2, BMP-4, CTGF, GDNF, NGF, FGF2, and PDGF-BB at the dose of 10 ng/ml for 24 hours, and ACTA2 ( A ), TAGLN ( B ), COL1A1 ( C ), and FN1 ( D ) expression levels were analyzed. E-H , Müller glial cells were treated for 24 hours with the reaction mixture of 10 ng/ml TGF-β1 preincubated with anti-TGF-β1 neutralizing antibody or control normal IgG at the dose of 200 ng/ml for 15 minutes, and ACTA2 ( E ), TAGLN ( F ), COL1A1 ( G ), and FN1 ( H ) gene expression levels were analyzed. n = 5–6 per group, * p

    Journal: Scientific Reports

    Article Title: TGF-β-SNAIL axis induces Müller glial-mesenchymal transition in the pathogenesis of idiopathic epiretinal membrane

    doi: 10.1038/s41598-018-36917-9

    Figure Lengend Snippet: TGF-β1 and TGF-β2, but not other pro-fibrotic cytokines, exclusively induces the expression of EMT markers in Müller glial cells. ( A–D ) Müller glial cells were treated with TGF-β1, TGF-β2, BMP-4, CTGF, GDNF, NGF, FGF2, and PDGF-BB at the dose of 10 ng/ml for 24 hours, and ACTA2 ( A ), TAGLN ( B ), COL1A1 ( C ), and FN1 ( D ) expression levels were analyzed. E-H , Müller glial cells were treated for 24 hours with the reaction mixture of 10 ng/ml TGF-β1 preincubated with anti-TGF-β1 neutralizing antibody or control normal IgG at the dose of 200 ng/ml for 15 minutes, and ACTA2 ( E ), TAGLN ( F ), COL1A1 ( G ), and FN1 ( H ) gene expression levels were analyzed. n = 5–6 per group, * p

    Article Snippet: Mouse anti-TGF-β1 neutralizing antibody and normal mouse IgG were purchased from R & D Systems (Minneapolis, MN) and preincubated at the dose of 200 ng/ml for 15 minutes with 10 ng/ml TGF-β1.

    Techniques: Expressing