salmon sperm dna  (Thermo Fisher)


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    Name:
    Salmon Sperm DNA
    Description:
    Sheared Salmon Sperm DNA is a very effective blocking agent when used in Northern prehybridization and hybridization buffers at a concentration of 100 200 µg mL Using this productThis sheared salmon sperm DNA has been treated with Proteinase K to remove any contaminating nucleases followed by organic extraction with phenol chloroform and ethanol precipitation Salmon Sperm DNA is rigorously tested for RNase and DNase contamination and is suspended in nuclease free water in a 1 mL tube at a concentration of 10 mg mL 1 mL per tube
    Catalog Number:
    am9680
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|Northern Blotting|Southern Blotting|Nucleic Acid Gel Electrophoresis & Blotting|Gene Expression Analysis & Genotyping|Microarray Hybridization & General Reagents
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher salmon sperm dna
    Activation of <t>p53</t> by pTpT. Cultures of H1299 cells were transfected with either control vector or the p53 expression vector, treated with pTpT and processed for the electromobility shift assay as described in Materials and Methods . Lane 1, extract from cells transfected with the p53 expression vector. Lane 2, cells were transfected with the p53 expression vector and incubated with 100 μM pTpT. Lane 3, same as lane 2 but the binding reaction contained 0.1 μg mAb421. Lane 4, same as lane 3 but with a 100-fold excess (10 pmol) unlabeled wild-type p53 consensus sequence. Lane 5, same as lane 3 but with a 100-fold express (10 pmol) unlabeled mutant p53 consensus sequence. The consensus sequence <t>DNA/p53</t> complex (C.S. DNA/p53) and the supershifted complex (C.S. DNA/p53/Ab421) are indicated by arrows.
    Sheared Salmon Sperm DNA is a very effective blocking agent when used in Northern prehybridization and hybridization buffers at a concentration of 100 200 µg mL Using this productThis sheared salmon sperm DNA has been treated with Proteinase K to remove any contaminating nucleases followed by organic extraction with phenol chloroform and ethanol precipitation Salmon Sperm DNA is rigorously tested for RNase and DNase contamination and is suspended in nuclease free water in a 1 mL tube at a concentration of 10 mg mL 1 mL per tube
    https://www.bioz.com/result/salmon sperm dna/product/Thermo Fisher
    Average 99 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    salmon sperm dna - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response"

    Article Title: Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response

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

    doi:

    Activation of p53 by pTpT. Cultures of H1299 cells were transfected with either control vector or the p53 expression vector, treated with pTpT and processed for the electromobility shift assay as described in Materials and Methods . Lane 1, extract from cells transfected with the p53 expression vector. Lane 2, cells were transfected with the p53 expression vector and incubated with 100 μM pTpT. Lane 3, same as lane 2 but the binding reaction contained 0.1 μg mAb421. Lane 4, same as lane 3 but with a 100-fold excess (10 pmol) unlabeled wild-type p53 consensus sequence. Lane 5, same as lane 3 but with a 100-fold express (10 pmol) unlabeled mutant p53 consensus sequence. The consensus sequence DNA/p53 complex (C.S. DNA/p53) and the supershifted complex (C.S. DNA/p53/Ab421) are indicated by arrows.
    Figure Legend Snippet: Activation of p53 by pTpT. Cultures of H1299 cells were transfected with either control vector or the p53 expression vector, treated with pTpT and processed for the electromobility shift assay as described in Materials and Methods . Lane 1, extract from cells transfected with the p53 expression vector. Lane 2, cells were transfected with the p53 expression vector and incubated with 100 μM pTpT. Lane 3, same as lane 2 but the binding reaction contained 0.1 μg mAb421. Lane 4, same as lane 3 but with a 100-fold excess (10 pmol) unlabeled wild-type p53 consensus sequence. Lane 5, same as lane 3 but with a 100-fold express (10 pmol) unlabeled mutant p53 consensus sequence. The consensus sequence DNA/p53 complex (C.S. DNA/p53) and the supershifted complex (C.S. DNA/p53/Ab421) are indicated by arrows.

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Electro Mobility Shift Assay, Incubation, Binding Assay, Sequencing, Mutagenesis

    2) Product Images from "Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR"

    Article Title: Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky535

    The long Pabpn1 3′UTR contains putative conserved cis- regulatory elements. ( A ) Schematic of the Pabpn1 transcript including a 5′ untranslated region (5′UTR), the coding DNA sequence ( Pabpn1 CDS), which encodes the PABPN1 open reading frame, and a 3′ untranslated region (3′UTR) which contains two polyadenylation signals (PASI, PASII) and multiple putative AU-rich elements (ARE1, ARE2, ARE3, ARE4). ( B ) Schematic of qRT-PCR strategy using primers that recognize the coding DNA sequence (CDS primers) and distal (distal primers) regions to determine which polyadenylation site (PASI or PASII) is utilized in C2C12 myoblasts (MB) and C2C12 myotubes (MT). ( C ) qRT-PCR was used to quantify Pabpn1 levels using primers that recognize the Pabpn1 CDS or the distal Pabpn1 3′UTR. The levels of distal 3′UTR-containing transcripts is calculated relative to CDS-containing transcripts and normalized to Gapdh . As described in Materials and Methods, these data are presented as fold change relative to C2C12 myoblasts, for which the average value was set to 1.0. Data are mean ± SEM of n = 3 samples per cell type. ( D ) Representative northern blot using a radiolabeled probe recognizing both short (1.4 kb) and long (2.1kb) forms of the Pabpn1 ), respectively, assessing PAS usage in multiple mouse tissues and cell lines including C2C12 myoblasts (MB) and C2C12 myotubes (MT) that have been differentiated for 10 days. Because testis tissue contains high levels of predominantly short Pabpn1 transcript, this sample was underloaded to avoid strong signal. The 18S (1.9 kb) rRNA serves as a loading control. Data are representative of n = 4 independent biological replicates for C2C12 myotubes and n = 4 technical replicates for C2C12 myoblasts. ( E ) Quantification of Pabpn1 transcript levels from four independent northern blots of RNA prepared from C2C12 myoblasts (MB) and C2C12 myotubes (MT) was performed as described in Materials and Methods. Data were normalized to 18S rRNA as the loading control and are presented as fold change in Pabpn1 transcript levels relative to myoblast, which was set to 1.0. Data are mean ± SEM of n = 4 (* P
    Figure Legend Snippet: The long Pabpn1 3′UTR contains putative conserved cis- regulatory elements. ( A ) Schematic of the Pabpn1 transcript including a 5′ untranslated region (5′UTR), the coding DNA sequence ( Pabpn1 CDS), which encodes the PABPN1 open reading frame, and a 3′ untranslated region (3′UTR) which contains two polyadenylation signals (PASI, PASII) and multiple putative AU-rich elements (ARE1, ARE2, ARE3, ARE4). ( B ) Schematic of qRT-PCR strategy using primers that recognize the coding DNA sequence (CDS primers) and distal (distal primers) regions to determine which polyadenylation site (PASI or PASII) is utilized in C2C12 myoblasts (MB) and C2C12 myotubes (MT). ( C ) qRT-PCR was used to quantify Pabpn1 levels using primers that recognize the Pabpn1 CDS or the distal Pabpn1 3′UTR. The levels of distal 3′UTR-containing transcripts is calculated relative to CDS-containing transcripts and normalized to Gapdh . As described in Materials and Methods, these data are presented as fold change relative to C2C12 myoblasts, for which the average value was set to 1.0. Data are mean ± SEM of n = 3 samples per cell type. ( D ) Representative northern blot using a radiolabeled probe recognizing both short (1.4 kb) and long (2.1kb) forms of the Pabpn1 ), respectively, assessing PAS usage in multiple mouse tissues and cell lines including C2C12 myoblasts (MB) and C2C12 myotubes (MT) that have been differentiated for 10 days. Because testis tissue contains high levels of predominantly short Pabpn1 transcript, this sample was underloaded to avoid strong signal. The 18S (1.9 kb) rRNA serves as a loading control. Data are representative of n = 4 independent biological replicates for C2C12 myotubes and n = 4 technical replicates for C2C12 myoblasts. ( E ) Quantification of Pabpn1 transcript levels from four independent northern blots of RNA prepared from C2C12 myoblasts (MB) and C2C12 myotubes (MT) was performed as described in Materials and Methods. Data were normalized to 18S rRNA as the loading control and are presented as fold change in Pabpn1 transcript levels relative to myoblast, which was set to 1.0. Data are mean ± SEM of n = 4 (* P

    Techniques Used: Sequencing, Quantitative RT-PCR, Northern Blot

    3) Product Images from "Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity"

    Article Title: Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    Journal: Scientific Reports

    doi: 10.1038/srep38065

    In vitro and in vivo assays to validate VSR activity. ( a ) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. ( b and c ) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. ( d ) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] ATP. 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. ( e )Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6 7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg 2 μg) of uncompetitive inhibitor. Salmon sperm DNA (2 μg) was used as non specific inhibitor in all binding reactions.
    Figure Legend Snippet: In vitro and in vivo assays to validate VSR activity. ( a ) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. ( b and c ) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. ( d ) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] ATP. 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. ( e )Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6 7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg 2 μg) of uncompetitive inhibitor. Salmon sperm DNA (2 μg) was used as non specific inhibitor in all binding reactions.

    Techniques Used: In Vitro, In Vivo, Activity Assay, Western Blot, Transfection, Transgenic Assay, Stable Transfection, Expressing, Positive Control, Negative Control, Northern Blot, Isolation, Electrophoretic Mobility Shift Assay, Labeling, shRNA, Binding Assay

    4) Product Images from "Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress"

    Article Title: Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003767

    Oxidative stress-mediated tRNA cleavage in HeLa cells. (A) Minor fraction tiRNAs are generated by longer exposure to 500 µM arsenite ( > 30 min). The numbers on the left denote the DNA ladder in nt. (B) Arsenite alters the integrity of the 3′-CCA end of full-length tRNAs in a dose-dependent manner. The amount of tRNAs with intact CCA ends was analyzed by their ability to ligate to a fluorescently labeled oligonucleotide (schematic inset) which forms a loop and binds only intact 3′-CCA ends (upper two gels). The intensity of tRNAs with intact CCA termini was quantified from the gels, normalized to the total tRNA amount at each time point and presented as relative values ± SEM (from three independent experiments) to the amount of initial, untreated sample which was set as 1.0. The amount of the total tRNA remained almost unchanged when cells were exposed to arsenite (500 µM) and visualized by SYBR Green (bottom gel marked as total tRNA). (C) Increase of the cellular concentration of angiogenin alters the 3′-CCA integrity of tRNAs. Angiogenin was upregulated by ectopic expression under the control of a CMV promoter for 8 h (+angiogenin). The sample representing arsenite stress (+arsenite) corresponds to the 60-min data point at 500 µM arsenite in panel (B) and is used for comparison. The intensity of tRNAs with intact CCA termini was the quantified as described for panel (B). ** for p
    Figure Legend Snippet: Oxidative stress-mediated tRNA cleavage in HeLa cells. (A) Minor fraction tiRNAs are generated by longer exposure to 500 µM arsenite ( > 30 min). The numbers on the left denote the DNA ladder in nt. (B) Arsenite alters the integrity of the 3′-CCA end of full-length tRNAs in a dose-dependent manner. The amount of tRNAs with intact CCA ends was analyzed by their ability to ligate to a fluorescently labeled oligonucleotide (schematic inset) which forms a loop and binds only intact 3′-CCA ends (upper two gels). The intensity of tRNAs with intact CCA termini was quantified from the gels, normalized to the total tRNA amount at each time point and presented as relative values ± SEM (from three independent experiments) to the amount of initial, untreated sample which was set as 1.0. The amount of the total tRNA remained almost unchanged when cells were exposed to arsenite (500 µM) and visualized by SYBR Green (bottom gel marked as total tRNA). (C) Increase of the cellular concentration of angiogenin alters the 3′-CCA integrity of tRNAs. Angiogenin was upregulated by ectopic expression under the control of a CMV promoter for 8 h (+angiogenin). The sample representing arsenite stress (+arsenite) corresponds to the 60-min data point at 500 µM arsenite in panel (B) and is used for comparison. The intensity of tRNAs with intact CCA termini was the quantified as described for panel (B). ** for p

    Techniques Used: Generated, Labeling, SYBR Green Assay, Concentration Assay, Expressing

    The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro . (A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.
    Figure Legend Snippet: The CCA sequence at the 3′-termini of all tRNAs is first cleaved by angiogenin in vitro . (A) Angiogenin (1 µM) digestion of total HeLa tRNA radioactively labeled at either 5′- or 3′-end. (B) 3′-radioactively labeled HeLa tRNAs treated with 1 µM angiogenin for different times and their intact 3′-termini were visualized with tRNA macroarrays. Only tRNAs (or fragments of them) with intact 3′-ends are visible on the microarrays. Two exemplary tRNAs (Ala-IGC, green and Gln-yTG, red) are marked. Probes for each tRNA are arranged in clusters of six replicates. (C) Analysis of the integrity of the 3′-CCA end of full-length tRNAs with the specific oligonucleotide-ligation approach after angiogenin (0.2 µM) treatment for various times. The gel was visualized with SYBR Green. Note, to better resolve the kinetics of cleavage we decreased the concentration of angiogenin to 0.2 µM; thus the time points here are not directly comparable with the time points in panels A, B. The numbers on the left denote the DNA ladder in nt. Multiple bands for tiRNAs, full-length tRNAs, ligated and unligated tRNAs are detected (panels A and C) due to the natural variations in tRNAs length.

    Techniques Used: Sequencing, In Vitro, Labeling, Ligation, SYBR Green Assay, Concentration Assay

    Human CCA-adding enzyme is able to repair damaged CCA ends of tRNAs. (A) Total HeLa tRNAs and (B) internally radioactively labeled yeast tRNA Phe were incubated successively with angiogenin and human CCA-adding enzyme. Subsequently to the angiogenin treatment (4 h), T4 polynucleotide kinase (PNK) (45 min) was added which converts the 2′,3′-cyclophosphate ends [42] generated by the angiogenin cleavage to free 3′OH. After purification tRNAs were subjected to treatment with the CCA-adding enzyme (30 min). The 3′-CCA end integrity of the HeLa tRNAs was determined with the fluorescent oligonucleotide ( Figure 1A , schematic inset). tRNA Phe lacking the terminal 3′-adenosine (tRNA Phe CC) served as a control. The numbers on the left denote the DNA ladder in nt.
    Figure Legend Snippet: Human CCA-adding enzyme is able to repair damaged CCA ends of tRNAs. (A) Total HeLa tRNAs and (B) internally radioactively labeled yeast tRNA Phe were incubated successively with angiogenin and human CCA-adding enzyme. Subsequently to the angiogenin treatment (4 h), T4 polynucleotide kinase (PNK) (45 min) was added which converts the 2′,3′-cyclophosphate ends [42] generated by the angiogenin cleavage to free 3′OH. After purification tRNAs were subjected to treatment with the CCA-adding enzyme (30 min). The 3′-CCA end integrity of the HeLa tRNAs was determined with the fluorescent oligonucleotide ( Figure 1A , schematic inset). tRNA Phe lacking the terminal 3′-adenosine (tRNA Phe CC) served as a control. The numbers on the left denote the DNA ladder in nt.

    Techniques Used: Labeling, Incubation, Generated, Purification

    5) Product Images from "Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis"

    Article Title: Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis

    Journal: Nature genetics

    doi: 10.1038/71732

    Fluorescence in situ hybridization (FISH). AIPL1 -containing bacterial artificial chromosome (BAC), shown in red, hybridizes to 17p13.1, consistent with placement of AIPL1 in the Stanford G3 radiation hybrid panel. These data refute the original placement of AIPL1 to 17p13.3 by placement in the GeneBridge 4.0 radiation hybrid panel. Chromosome 17 α-satellite DNA is indicated (green).
    Figure Legend Snippet: Fluorescence in situ hybridization (FISH). AIPL1 -containing bacterial artificial chromosome (BAC), shown in red, hybridizes to 17p13.1, consistent with placement of AIPL1 in the Stanford G3 radiation hybrid panel. These data refute the original placement of AIPL1 to 17p13.3 by placement in the GeneBridge 4.0 radiation hybrid panel. Chromosome 17 α-satellite DNA is indicated (green).

    Techniques Used: Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, BAC Assay

    6) Product Images from "Gemfibrozil, a Lipid-lowering Drug, Induces Suppressor of Cytokine Signaling 3 in Glial Cells"

    Article Title: Gemfibrozil, a Lipid-lowering Drug, Induces Suppressor of Cytokine Signaling 3 in Glial Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.346932

    Gemfibrozil induces the recruitment of KLF4 to the distal KLF4-binding site on the Socs3 promoter. A, DNA sequence of the Socs3 promoter region containing the KLF4-binding sites with positions of the primers used for the ChIP analysis. Mouse BV-2 microglial
    Figure Legend Snippet: Gemfibrozil induces the recruitment of KLF4 to the distal KLF4-binding site on the Socs3 promoter. A, DNA sequence of the Socs3 promoter region containing the KLF4-binding sites with positions of the primers used for the ChIP analysis. Mouse BV-2 microglial

    Techniques Used: Binding Assay, Sequencing, Chromatin Immunoprecipitation

    7) Product Images from "DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides"

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051905

    DNA bound to M. xanthus EPS at different pHs. The binding percentages of wild-type DK1622 chromosomal DNA (left) and commercial salmon sperm DNA (right) to isolated EPS were determined at different pHs, and the average ± SD is plotted. On the x-coordinate, ‘DNA’ represents different DNA samples, ‘EPS’ represents isolated M. xanthus EPS and ‘Cell*’ represents SW504 cells, which were added to the test system.
    Figure Legend Snippet: DNA bound to M. xanthus EPS at different pHs. The binding percentages of wild-type DK1622 chromosomal DNA (left) and commercial salmon sperm DNA (right) to isolated EPS were determined at different pHs, and the average ± SD is plotted. On the x-coordinate, ‘DNA’ represents different DNA samples, ‘EPS’ represents isolated M. xanthus EPS and ‘Cell*’ represents SW504 cells, which were added to the test system.

    Techniques Used: Binding Assay, Isolation

    8) Product Images from "Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach"

    Article Title: Plasma Exosome Profiling of Cancer Patients by a Next Generation Systems Biology Approach

    Journal: Scientific Reports

    doi: 10.1038/srep42741

    Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).
    Figure Legend Snippet: Oligonucleotides identified by ADAPT reveal aptamer-like characteristics. ( a ) Filter retention analysis of C1Q-binding by the ssODNs H1 (dark grey columns) and H11 (pale grey columns) at indicated C1Q concentrations. As a control, the reverse complement of H1, H1RC, was used (black columns). ( b ) ELONA analysis of C1Q-binding by the ssODNs H11 (circles) at indicated concentrations and a fixed C1Q concentration (0.625 nM). As a control, the reverse complement of H11, H11RC, was used (squares). “No aptamer” control (triangles) shows low background binding of detector Streptavidin-HRP. H11 specifically binds C1Q (estimated K D around 40 nM). ( c ) PAGE analysis of PEG-precipitated and ssODN-associated proteins pulled-down with L2. Lane 1: Molecular weight marker; lane 2: Input library L2; lane 3: Fraction pulled down by biotinylated L2; lane 4: Fraction pulled down by non-biotinylated L2; lane 5: Fraction found in the absence of DNA library; red arrows indicate the ssODN library; yellow arrows indicate protein bands cut out and analysed by LC-MS/MS; black arrows indicate streptavidin monomers leaking from beads. ( d ) Four-way Venn diagram of proteins detected by LC-MS/MS from Unfractionated plasma, PEG-precipitated plasma, PEG-precipitated plasma in presence of L0 or L2, respectively, purified by streptavidin magnetic beads (background-subtracted; i.e. biotinylated minus non-biotinylated libraries). ( e ) Gene ontology (GO) cellular component enrichment analysis of the subset of proteins associated with L0 (red bars) and L2 (grey bars) that show a p value of at least 6 × 10 −12 . Proteins listed are: 1 extracellular region part, 2 extracellular region, 3 extracellular exosome, 4 extracellular membrane-bounded organelle, 5 extracellular organelle, 6 extracellular vesicle, 7 membrane-bounded vesicle, 8 vesicle, 9 organelle, 10 membrane-bounded organelle, 11 extracellular space, 12 focal adhesion, 13 cell-substrate adherence junction, 14 cell-substrate junction, 15 adherence junction, 16 anchoring junction, 17 cellular component, 18 cell junction, 19 blood micro particle. Inset: 108 proteins pulled down by L2 (inset, 96 + 12), cut from the gel shown in ( c ), and analysed by LC-MS/MS, 13 proteins unique to background-subtracted L0 (inset, 13), and 12 overlapping proteins (inset, 12).

    Techniques Used: Binding Assay, Concentration Assay, Polyacrylamide Gel Electrophoresis, Molecular Weight, Marker, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Magnetic Beads

    9) Product Images from "Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein"

    Article Title: Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-61

    Electromobility shift analysis of IsaB . A . Purified recombinant IsaB was analyzed by EMSA assay using a fluorescently labeled RNA probe. IsaB shifted the RNA probe in a concentration dependent manner. A. Lane 1, RNA probe alone Lane 2, RNA probe + 3.84 nmol of IsaB, Lane 3, RNA probe + 1.92 nmol of IsaB, Lane 4, RNA probe + 960 pmol of IsaB, Lane 5, RNA probe + 480 pmol of IsaB, Lane 6, RNA probe + 240 pmol of IsaB. At the highest concentrations of IsaB, the RNA probe appeared to aggregate within the wells, while at lower concentrations of IsaB (lanes 4–6) a fraction of the RNA shifted (arrow) but some RNA still remained in the wells. B . Effect of salmon sperm DNA on shift; 480 pmol IsaB and 270 pmol labeled. RNA were added to each reaction. Lane 1, RNA probe alone, Lane 2, IsaB, + RNA probe, Lane 3, IsaB + RNA probe and 1.35 nmol unlabeled DNA, Lane 4, IsaB + RNA and 135 pmol unlabeled DNA, Lane 5, IsaB + RNA and 13.5 pmol unlabeled DNA, Lane 6, IsaB + RNA and 1.35 pmol unlabeled DNA.
    Figure Legend Snippet: Electromobility shift analysis of IsaB . A . Purified recombinant IsaB was analyzed by EMSA assay using a fluorescently labeled RNA probe. IsaB shifted the RNA probe in a concentration dependent manner. A. Lane 1, RNA probe alone Lane 2, RNA probe + 3.84 nmol of IsaB, Lane 3, RNA probe + 1.92 nmol of IsaB, Lane 4, RNA probe + 960 pmol of IsaB, Lane 5, RNA probe + 480 pmol of IsaB, Lane 6, RNA probe + 240 pmol of IsaB. At the highest concentrations of IsaB, the RNA probe appeared to aggregate within the wells, while at lower concentrations of IsaB (lanes 4–6) a fraction of the RNA shifted (arrow) but some RNA still remained in the wells. B . Effect of salmon sperm DNA on shift; 480 pmol IsaB and 270 pmol labeled. RNA were added to each reaction. Lane 1, RNA probe alone, Lane 2, IsaB, + RNA probe, Lane 3, IsaB + RNA probe and 1.35 nmol unlabeled DNA, Lane 4, IsaB + RNA and 135 pmol unlabeled DNA, Lane 5, IsaB + RNA and 13.5 pmol unlabeled DNA, Lane 6, IsaB + RNA and 1.35 pmol unlabeled DNA.

    Techniques Used: Purification, Recombinant, Labeling, Concentration Assay

    Competitive Electromobility shift analysis . EMSAs were performed with unlabeled competitors added to the reactions. 480 pmol IsaB and 270 pmol labeled RNA were included in each sample. Lane 1, labeled probe alone, Lane 2, IsaB + labeled RNA, Lane 3, IsaB + labeled RNA and 270 pmol unlabeled DNA, Lane 4, IsaB + labeled RNA and 270 pmol dNTPs, Lane 5, IsaB + labeled RNA and 270 pmol yeast tRNA.
    Figure Legend Snippet: Competitive Electromobility shift analysis . EMSAs were performed with unlabeled competitors added to the reactions. 480 pmol IsaB and 270 pmol labeled RNA were included in each sample. Lane 1, labeled probe alone, Lane 2, IsaB + labeled RNA, Lane 3, IsaB + labeled RNA and 270 pmol unlabeled DNA, Lane 4, IsaB + labeled RNA and 270 pmol dNTPs, Lane 5, IsaB + labeled RNA and 270 pmol yeast tRNA.

    Techniques Used: Labeling

    10) Product Images from "Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance ▿"

    Article Title: Evidence that Regulatory Protein MarA of Escherichia coli Represses rob by Steric Hindrance ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00103-10

    Results of gel shift experiment showing the effect of preincubation with MarA on initiation complex (RDM) formation by RNAP at activated promoter fumC . The 37°C assay was used. DNA and MarA were detected as described for Fig. .
    Figure Legend Snippet: Results of gel shift experiment showing the effect of preincubation with MarA on initiation complex (RDM) formation by RNAP at activated promoter fumC . The 37°C assay was used. DNA and MarA were detected as described for Fig. .

    Techniques Used: Electrophoretic Mobility Shift Assay

    11) Product Images from "SPATIAL ORGANIZATION OF TRANSCRIBED EUKARYOTIC GENES"

    Article Title: SPATIAL ORGANIZATION OF TRANSCRIBED EUKARYOTIC GENES

    Journal: bioRxiv

    doi: 10.1101/2020.05.20.106591

    TLs manifest transcription progression, including co-transcriptional splicing, and dynamically modify harboring chromosomal loci A , Successive labeling of Tg TLs with probes for 5’ and 3’ exons. A1, Distribution of 11 5’ exons ( green ) and 15 3’ exons ( red ) of the Tg gene chosen for RNA-FISH. A2,3, Schematics showing changes in the composition of nRNAs during transcription progression: nRNAs of the first half of a TL contain only “ green ” 5’ exons; in the second half they also include “ red ” 3’ exons (A2). Correspondingly, “ green ” exons label transcripts along the whole TL and “ red ” exons label only transcripts of the TL second half (A3). A4, Examples of Tg TLs labeling after RNA-FISH with probes described in A1. Probe for the 5’ exons labels the entire TL, whereas the probe for 3’ exons labels second half of the TL. Arrows point at the TL regions labeled with only 5’ probe. B , Sequential labeling of Tg TLs with genomic probes highlighting introns. B1, coverage of the Tg gene with two overlapping BACs, for 5’ ( green ) and 3’ ( red ) gene halves. B2,3, Schematics explaining changes in nRNA labeling with probes highlighting introns during transcription progression: “ green ” 5’ introns are gradually spliced out and replaced by “ red ” 3’ introns (B2). Correspondingly, “ green ” introns label transcripts of the first and “ red ” exons of the second TL halves with some overlap ( yellow , B3). B4, Example of Tg TLs after RNA-FISH with two BAC probes described in B1. BAC probes highlighting 5’ ( green ) and 3’ ( red ) introns sequentially label the TLs as a result of co-transcriptional splicing. In addition, since the 5’ probe includes 5’ exons, it also faintly labeled the second half of the loop ( arrows ). The region hybridized by both overlapping BACs is marked with arrowheads. ex , exons; int , introns. Black arrows indicate direction of transcription. Scale bars, 2 µm C , TLs formed by other long highly expressed genes exhibit co-transcriptional splicing. RNA-FISH with BAC probes encompassing 5’ ( green ) and 3’ ( red ) regions of the genes (see SI Table S3 for the probes). Projections of confocal sections through 1 - 2 µm . Scale bars, 2 µm D , Expressed genes expand from the harboring loci and separate their flanks. Distances between 5’ ( green ) and 3’ ( red ) flanking regions of the Tg (D1) and Ttn (D2) genes are larger in cells expressing ( left panels ) compared to control cells not expressing the genes ( right panels ), as shown by schematics at the bottom. Boxplots depicting the 3D distance between the flanking regions in expressing and not-expressing cells are shown on the right. The median inter-flank distance for Tg in thyrocytes is 2.3-fold larger than in control epithelial cells (703 nm versus 311 nm). The median inter-flank distance for Ttn in myotubes is 1.7-fold larger than in control myoblasts (1,104 nm versus 634 nm). FISH with simultaneous detection of DNA and RNA signals. Projections of confocal sections through 1 - 3 µm . Scale bars, 2 µm
    Figure Legend Snippet: TLs manifest transcription progression, including co-transcriptional splicing, and dynamically modify harboring chromosomal loci A , Successive labeling of Tg TLs with probes for 5’ and 3’ exons. A1, Distribution of 11 5’ exons ( green ) and 15 3’ exons ( red ) of the Tg gene chosen for RNA-FISH. A2,3, Schematics showing changes in the composition of nRNAs during transcription progression: nRNAs of the first half of a TL contain only “ green ” 5’ exons; in the second half they also include “ red ” 3’ exons (A2). Correspondingly, “ green ” exons label transcripts along the whole TL and “ red ” exons label only transcripts of the TL second half (A3). A4, Examples of Tg TLs labeling after RNA-FISH with probes described in A1. Probe for the 5’ exons labels the entire TL, whereas the probe for 3’ exons labels second half of the TL. Arrows point at the TL regions labeled with only 5’ probe. B , Sequential labeling of Tg TLs with genomic probes highlighting introns. B1, coverage of the Tg gene with two overlapping BACs, for 5’ ( green ) and 3’ ( red ) gene halves. B2,3, Schematics explaining changes in nRNA labeling with probes highlighting introns during transcription progression: “ green ” 5’ introns are gradually spliced out and replaced by “ red ” 3’ introns (B2). Correspondingly, “ green ” introns label transcripts of the first and “ red ” exons of the second TL halves with some overlap ( yellow , B3). B4, Example of Tg TLs after RNA-FISH with two BAC probes described in B1. BAC probes highlighting 5’ ( green ) and 3’ ( red ) introns sequentially label the TLs as a result of co-transcriptional splicing. In addition, since the 5’ probe includes 5’ exons, it also faintly labeled the second half of the loop ( arrows ). The region hybridized by both overlapping BACs is marked with arrowheads. ex , exons; int , introns. Black arrows indicate direction of transcription. Scale bars, 2 µm C , TLs formed by other long highly expressed genes exhibit co-transcriptional splicing. RNA-FISH with BAC probes encompassing 5’ ( green ) and 3’ ( red ) regions of the genes (see SI Table S3 for the probes). Projections of confocal sections through 1 - 2 µm . Scale bars, 2 µm D , Expressed genes expand from the harboring loci and separate their flanks. Distances between 5’ ( green ) and 3’ ( red ) flanking regions of the Tg (D1) and Ttn (D2) genes are larger in cells expressing ( left panels ) compared to control cells not expressing the genes ( right panels ), as shown by schematics at the bottom. Boxplots depicting the 3D distance between the flanking regions in expressing and not-expressing cells are shown on the right. The median inter-flank distance for Tg in thyrocytes is 2.3-fold larger than in control epithelial cells (703 nm versus 311 nm). The median inter-flank distance for Ttn in myotubes is 1.7-fold larger than in control myoblasts (1,104 nm versus 634 nm). FISH with simultaneous detection of DNA and RNA signals. Projections of confocal sections through 1 - 3 µm . Scale bars, 2 µm

    Techniques Used: Labeling, Fluorescence In Situ Hybridization, BAC Assay, Expressing

    12) Product Images from "PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells"

    Article Title: PYHIN1 regulates pro-inflammatory cytokine induction rather than innate immune DNA sensing in airway epithelial cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011400

    STING expression restores a cGAS-dependent response to DNA in A549 cells. A, 1 × 10 5 /ml A549 cells were transfected with IFNβ promoter luciferase reporter together with EV or increasing amounts of AIM2, IFI16, MNDA, PYHIN1, cGAS, STING, or DDX41 expression vectors. Wedges indicate increasing amount of the expression vectors (2, 10, 50 ng). Cells were lysed 24 h after transfection and assessed for reporter gene activity. Data are shown as relative fold-induction normalized to EV-only transfected cells. The data are mean ± S.D. of triplicate samples and are representative of three experiments. B, A549 cells (0.5 × 10 5 /ml) were transfected with 12.5 ng/ml control or cGAS siRNA 24 h and then again 48 h after cells were seeded. 24 h later cell lysates were generated and immunoblotted for cGAS and β-actin. Representative of two experiments. C, cells were treated with siRNA as in B , and 24 h later transfected with IFNβ promoter luciferase reporter together with EV or increasing amounts of STING expression vector. Wedge indicates increasing amount of STING expression vector (2, 10, 50 ng). Cells were lysed 24 h after transfection and assessed for reporter gene activity. ***, p
    Figure Legend Snippet: STING expression restores a cGAS-dependent response to DNA in A549 cells. A, 1 × 10 5 /ml A549 cells were transfected with IFNβ promoter luciferase reporter together with EV or increasing amounts of AIM2, IFI16, MNDA, PYHIN1, cGAS, STING, or DDX41 expression vectors. Wedges indicate increasing amount of the expression vectors (2, 10, 50 ng). Cells were lysed 24 h after transfection and assessed for reporter gene activity. Data are shown as relative fold-induction normalized to EV-only transfected cells. The data are mean ± S.D. of triplicate samples and are representative of three experiments. B, A549 cells (0.5 × 10 5 /ml) were transfected with 12.5 ng/ml control or cGAS siRNA 24 h and then again 48 h after cells were seeded. 24 h later cell lysates were generated and immunoblotted for cGAS and β-actin. Representative of two experiments. C, cells were treated with siRNA as in B , and 24 h later transfected with IFNβ promoter luciferase reporter together with EV or increasing amounts of STING expression vector. Wedge indicates increasing amount of STING expression vector (2, 10, 50 ng). Cells were lysed 24 h after transfection and assessed for reporter gene activity. ***, p

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Generated, Plasmid Preparation

    13) Product Images from "Routes to DNA Accessibility: Alternative Pathways for Nucleosome Unwinding"

    Article Title: Routes to DNA Accessibility: Alternative Pathways for Nucleosome Unwinding

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2014.05.042

    Cartoon of possible nucleosome states and their implications in vivo. DNA is black, the nucleosome octamer is shown as an H3-H4 tetramer in magenta, and the H2A-H2B dimer is in yellow. ( A ) Nucleosome interactions with various proteins. The circular
    Figure Legend Snippet: Cartoon of possible nucleosome states and their implications in vivo. DNA is black, the nucleosome octamer is shown as an H3-H4 tetramer in magenta, and the H2A-H2B dimer is in yellow. ( A ) Nucleosome interactions with various proteins. The circular

    Techniques Used: In Vivo

    Model of nucleosome unwinding. ( A ) Kinetic diagram of nucleosome unwinding and rewinding. DNA is shown in blue and the octamer is in red. In state 2, the DNA is wrapped nearly two full times around the octamer. In state 1, the outer turn is unwrapped
    Figure Legend Snippet: Model of nucleosome unwinding. ( A ) Kinetic diagram of nucleosome unwinding and rewinding. DNA is shown in blue and the octamer is in red. In state 2, the DNA is wrapped nearly two full times around the octamer. In state 1, the outer turn is unwrapped

    Techniques Used:

    14) Product Images from "Peroxisome proliferator‐activated receptor gamma (PPARγ) regulates lactase expression and activity in the gut"

    Article Title: Peroxisome proliferator‐activated receptor gamma (PPARγ) regulates lactase expression and activity in the gut

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201707795

    LCT genotyping of polymorphisms C/T 13910 and G/A 22018 for Caco‐2 cells DNA genomic fragments encompassing C/T 13910 and G/A 22018 nucleotides were amplified and digested as described in Matthews et al ( 2005 ). The digested PCR products were analyzed on agarose gel. Lanes 1 and 8 show molecular weight markers. Lanes 2 and 9 show non‐digested (ND) PCR products. Lanes 3 and 10 show the CC 13910 and GG 22018 homozygous lactose intolerance genotypes, respectively. Lanes 4 and 11 show the TT 13910 and AA 22018 homozygous lactase persistent genotypes, respectively. Lanes 5 and 12 show the CT 13910 and GA 22018 heterozygous lactase persistent genotypes, respectively. Lanes 6 and 13 show non‐digested PCR products from Caco‐2 cells. Lanes 7 and 14 show digested PCR products displaying LCT genotyping polymorphisms C/T 13910 and G/A 22018 for Caco‐2 cells. Migration profile reveals that Caco‐2 cells possess the CC 13910 (lane 7) and GG 22018 (lane 14) homozygous lactose intolerance genotypes.
    Figure Legend Snippet: LCT genotyping of polymorphisms C/T 13910 and G/A 22018 for Caco‐2 cells DNA genomic fragments encompassing C/T 13910 and G/A 22018 nucleotides were amplified and digested as described in Matthews et al ( 2005 ). The digested PCR products were analyzed on agarose gel. Lanes 1 and 8 show molecular weight markers. Lanes 2 and 9 show non‐digested (ND) PCR products. Lanes 3 and 10 show the CC 13910 and GG 22018 homozygous lactose intolerance genotypes, respectively. Lanes 4 and 11 show the TT 13910 and AA 22018 homozygous lactase persistent genotypes, respectively. Lanes 5 and 12 show the CT 13910 and GA 22018 heterozygous lactase persistent genotypes, respectively. Lanes 6 and 13 show non‐digested PCR products from Caco‐2 cells. Lanes 7 and 14 show digested PCR products displaying LCT genotyping polymorphisms C/T 13910 and G/A 22018 for Caco‐2 cells. Migration profile reveals that Caco‐2 cells possess the CC 13910 (lane 7) and GG 22018 (lane 14) homozygous lactose intolerance genotypes.

    Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Migration

    15) Product Images from "Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses"

    Article Title: Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses

    Journal: Scientific Reports

    doi: 10.1038/srep45458

    Validation of new NikR promoters and internal peaks by DNase I footprinting. ( A ) Radiolabeled P ureA , P hcpC , P mccB and P hopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆ nikR /ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. ( B ) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel). The same elements and information are reported as in panel A.
    Figure Legend Snippet: Validation of new NikR promoters and internal peaks by DNase I footprinting. ( A ) Radiolabeled P ureA , P hcpC , P mccB and P hopV DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel), before DNase I cleavage. On the right of each autoradiographic film, the G27 genomic coordinates of DNase I protected regions (black boxes) are reported, with position in brackets with respect to the transcriptional start site (TSS). Low affinity binding sites, if present, are shown as grey boxes surrounded by the same information. On the left, a schematic representation of the promoter is provided, with the TSS (+1, bent arrow) and the −10 region (black box). The position of the consensus sequence is reported with violet boxes, corresponding to the two conserved hemi-operator pentamers linked by a black line (15 nt spacer). In the middle panels a scheme of the corresponding transcriptional unit is shown, together with the normalized tag densities obtained from the ChIP-seq experiments (wt/ni+ in yellow, wt/ni− in orange and ∆ nikR /ni+, negative control in green), the predicted peak extension by Homer2 and the DNaseI protected regions. Representation scales of ChIP-seq tracks are indicated on the left in brackets. In the bottom panels, the RNA-seq strand specific tracks of the corresponding genomic locus are visualized for wt/ni+ and wt/ni− samples (plus strand in blue, minus strand in red). P* indicates coordinates mapping on the pHPG27 plasmid. ( B ) Radiolabeled dapD, exsB, fecD and pcrA DNA probes were mixed with 0, 9.7, 29, 97 and 290 nM of NikR tetramers, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel). The same elements and information are reported as in panel A.

    Techniques Used: Footprinting, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Negative Control, RNA Sequencing Assay, Plasmid Preparation

    Validation of the new NikR-dependent nickel-regulated ncRNAs. Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆ nikR /ni− and ∆ nikR /ni+ conditions. ( A ) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). ( B ) Primer extension analysis of the Nrr2 transcript. ( C ) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 ( A ), Nrr2 ( B ) and IsoB ( C ) DNA probes, mixed with 0, 9.7 (only panel B), 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel). Uncropped blots and gels are provided in the Supplementary Information . Legends and symbols as in Fig. 2 .
    Figure Legend Snippet: Validation of the new NikR-dependent nickel-regulated ncRNAs. Top panels: transcriptional analysis of ncRNAs in wt/ni−, wt/ni+, ∆ nikR /ni− and ∆ nikR /ni+ conditions. ( A ) Northern blot of Nrr1 (left) and quantitative RT-qPCR of its transcript levels (right) (see legend 2D for details). ( B ) Primer extension analysis of the Nrr2 transcript. ( C ) Northern blot of the IsoB transcript. Middle panels: DNase I footprinting of radiolabeled Nrr1 ( A ), Nrr2 ( B ) and IsoB ( C ) DNA probes, mixed with 0, 9.7 (only panel B), 29, 97 and 290 nM of the NikR tetramer, without nickel (left side of each panel) or with the addition of 150 μM NiSO 4 (right side of each panel). Uncropped blots and gels are provided in the Supplementary Information . Legends and symbols as in Fig. 2 .

    Techniques Used: Northern Blot, Quantitative RT-PCR, Footprinting

    16) Product Images from "Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor-DNA Interactions"

    Article Title: Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor-DNA Interactions

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000449

    An overview of Microarray profiling of phage-display selection technology. (A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.
    Figure Legend Snippet: An overview of Microarray profiling of phage-display selection technology. (A) 1–3 kb fragments of yeast genomic DNA are cloned into T7 bacteriophage to create a translational fusion between the capsid protein and the peptide sequence encoded by the insert. (B) The library of phage are exposed to immobilized target DNA molecules and non-binding phage are washed away. Bound phage are eluted, amplified in liquid culture, and the process is repeated over multiple rounds. The sequence content of the enriched phage population is determined by PCR amplification of the inserts, labeling, and hybridization to a yeast ORF microarray.

    Techniques Used: Microarray, Selection, Clone Assay, Sequencing, Binding Assay, Amplification, Polymerase Chain Reaction, Labeling, Hybridization

    17) Product Images from "Dual Functions of ?-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei"

    Article Title: Dual Functions of ?-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00269-12

    Localization and subcellular fractionation of α-KDE2-PTP. A partial α-KDE2 sequence was ligated into the C-terminal PTP expression vector (pC-PTP-NEO) and transfected into BF and PF T. brucei . (A) Northern analysis of α-KDE2-PTP transcript expression in PF and BF developmental stages. RNA from 5 × 10 6 cells was evaluated with radiolabeled probes against the α-KDE2 sequence. EtBr stain for rRNAs is shown for each lane. (B) Cellular localization of α-KDE2-PTP by immunofluorescence microscopy. α-KDE2-PTP PF and BF cells were stained for DNA with DAPI, mitochondria with MitoTracker, and α-KDE2-PTP with antibodies against the protein C epitope. (C) Subcellular fractionation of procyclic-form α-KDE2-PTP cells. Lysates from total PF-667 (WT) cells and fractionated PF α-KDE2-PTP cells (TC) were resolved by SDS-PAGE and analyzed by Western blotting using antibodies against HSP-70 (cytosolic marker), MTP-70 (mitochondrial matrix marker), ISP (mitochondrial membrane marker), and protein A epitope (α-KDE2-PTP). (D) Subcellular fractionation of BF α-KDE2-PTP cells. Lysates from total BF-667 (WT) cells and BF α-KDE2-PTP cells (TC) were fractionated and analyzed as for panel C with TAO as the mitochondrial membrane marker. The positions of the nucleus (n), kDNA (k), heat shock protein 70 (HSP-70), mitochondrial heat shock protein 70 (MTP-70), ISP, and TAO are indicated. Abbreviation are used for cells and organelle fractions in panels C and D: total cell protein for nontransfected (WT) and α-KDE2-PTP transfected (TC) T. brucei 667, cytosolic protein (CY), total mitochondrial protein (TM), mitochondrial matrix protein (MA), and mitochondrial membrane protein (ME).
    Figure Legend Snippet: Localization and subcellular fractionation of α-KDE2-PTP. A partial α-KDE2 sequence was ligated into the C-terminal PTP expression vector (pC-PTP-NEO) and transfected into BF and PF T. brucei . (A) Northern analysis of α-KDE2-PTP transcript expression in PF and BF developmental stages. RNA from 5 × 10 6 cells was evaluated with radiolabeled probes against the α-KDE2 sequence. EtBr stain for rRNAs is shown for each lane. (B) Cellular localization of α-KDE2-PTP by immunofluorescence microscopy. α-KDE2-PTP PF and BF cells were stained for DNA with DAPI, mitochondria with MitoTracker, and α-KDE2-PTP with antibodies against the protein C epitope. (C) Subcellular fractionation of procyclic-form α-KDE2-PTP cells. Lysates from total PF-667 (WT) cells and fractionated PF α-KDE2-PTP cells (TC) were resolved by SDS-PAGE and analyzed by Western blotting using antibodies against HSP-70 (cytosolic marker), MTP-70 (mitochondrial matrix marker), ISP (mitochondrial membrane marker), and protein A epitope (α-KDE2-PTP). (D) Subcellular fractionation of BF α-KDE2-PTP cells. Lysates from total BF-667 (WT) cells and BF α-KDE2-PTP cells (TC) were fractionated and analyzed as for panel C with TAO as the mitochondrial membrane marker. The positions of the nucleus (n), kDNA (k), heat shock protein 70 (HSP-70), mitochondrial heat shock protein 70 (MTP-70), ISP, and TAO are indicated. Abbreviation are used for cells and organelle fractions in panels C and D: total cell protein for nontransfected (WT) and α-KDE2-PTP transfected (TC) T. brucei 667, cytosolic protein (CY), total mitochondrial protein (TM), mitochondrial matrix protein (MA), and mitochondrial membrane protein (ME).

    Techniques Used: Fractionation, Sequencing, Expressing, Plasmid Preparation, Transfection, Northern Blot, Staining, Immunofluorescence, Microscopy, SDS Page, Western Blot, Marker

    18) Product Images from "IHF Is Required for the Transcriptional Regulation of the Desulfovibrio vulgaris Hildenborough orp Operons"

    Article Title: IHF Is Required for the Transcriptional Regulation of the Desulfovibrio vulgaris Hildenborough orp Operons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086507

    Sequence analyses of the orp1 and orp2 promoter regions. (A) The σ 54 and σ 70 promoters of the ORP system are indicated by bent arrows. The solid-line boxes indicate the palindromic DVU2106-binding sites. The dashed boxes represent the three putative IHF-binding sequences identified by comparison to the consensus sequence of the E.coli IHF-binding site (5′-WATCARxxxxTTR-3′). (B) DNA sequence alignment between the consensus sequence of E.coli IHF-binding site and each putative IHF-binding sequence of orp1 and orp2 promoter regions. The sequence identity between the consensus sequence of the E.coli IHF-binding site and the different orp putative IHF-binding site are indicated in bold.
    Figure Legend Snippet: Sequence analyses of the orp1 and orp2 promoter regions. (A) The σ 54 and σ 70 promoters of the ORP system are indicated by bent arrows. The solid-line boxes indicate the palindromic DVU2106-binding sites. The dashed boxes represent the three putative IHF-binding sequences identified by comparison to the consensus sequence of the E.coli IHF-binding site (5′-WATCARxxxxTTR-3′). (B) DNA sequence alignment between the consensus sequence of E.coli IHF-binding site and each putative IHF-binding sequence of orp1 and orp2 promoter regions. The sequence identity between the consensus sequence of the E.coli IHF-binding site and the different orp putative IHF-binding site are indicated in bold.

    Techniques Used: Sequencing, Binding Assay, Immunohistofluorescence

    19) Product Images from "A Small Loop in the Capsid Protein of Moloney Murine Leukemia Virus Controls Assembly of Spherical Cores"

    Article Title: A Small Loop in the Capsid Protein of Moloney Murine Leukemia Virus Controls Assembly of Spherical Cores

    Journal: Journal of Virology

    doi: 10.1128/JVI.80.6.2884-2893.2006

    Mixed virions are infectious. (A) The ability of mixed virions to infect cells was assayed using the viral spreading assay. Culture medium was collected after cotransfection with WT and mutant proviral DNAs in the indicated ratios. Equal amounts of released virus were used to inoculate naïve Rat2 cells. RT activity was measured in supernatants harvested 36 h after inoculation. (B) PCR using low-molecular-weight DNA from infected Rat2 cells. PCR primers flanked the region containing the insertional mutations, producing a 243-bp product from WT viral DNA and 279 bp from the mutant. Template DNAs consisted of mutant and WT proviral plasmid DNA (controls in lanes 1 and 2) and low-molecular-weight DNA from Rat2 cells infected with mutant virions (lane 3), mixed virions (cotransfections were done with equal amounts of WT and 1970 proviral DNA; lane 4), and WT virions (lane 5). The WT resulted in a PCR product of the expected size (lower arrow), while 1970 alone resulted in no PCR product, since it did not make virions. Mixed virions resulted in a slower-migrating band corresponding to the mutant product and a faster one for WT (doublet). PCR products resulting from amplification of rat mitochondrial DNA (bottom gel in panel B) indicated that nearly equal amounts of template were used in the analysis. (C) Rat2 cell lysates were harvested 36 h after inoculation with mixed virions and analyzed by Western blotting using anti-CA polyclonal serum. Arrows indicate WT and mutant CA protein. The same blot reprobed with antitubulin antibodies is shown below to indicate equal loading of lanes on the gel.
    Figure Legend Snippet: Mixed virions are infectious. (A) The ability of mixed virions to infect cells was assayed using the viral spreading assay. Culture medium was collected after cotransfection with WT and mutant proviral DNAs in the indicated ratios. Equal amounts of released virus were used to inoculate naïve Rat2 cells. RT activity was measured in supernatants harvested 36 h after inoculation. (B) PCR using low-molecular-weight DNA from infected Rat2 cells. PCR primers flanked the region containing the insertional mutations, producing a 243-bp product from WT viral DNA and 279 bp from the mutant. Template DNAs consisted of mutant and WT proviral plasmid DNA (controls in lanes 1 and 2) and low-molecular-weight DNA from Rat2 cells infected with mutant virions (lane 3), mixed virions (cotransfections were done with equal amounts of WT and 1970 proviral DNA; lane 4), and WT virions (lane 5). The WT resulted in a PCR product of the expected size (lower arrow), while 1970 alone resulted in no PCR product, since it did not make virions. Mixed virions resulted in a slower-migrating band corresponding to the mutant product and a faster one for WT (doublet). PCR products resulting from amplification of rat mitochondrial DNA (bottom gel in panel B) indicated that nearly equal amounts of template were used in the analysis. (C) Rat2 cell lysates were harvested 36 h after inoculation with mixed virions and analyzed by Western blotting using anti-CA polyclonal serum. Arrows indicate WT and mutant CA protein. The same blot reprobed with antitubulin antibodies is shown below to indicate equal loading of lanes on the gel.

    Techniques Used: Cotransfection, Mutagenesis, Activity Assay, Polymerase Chain Reaction, Molecular Weight, Infection, Plasmid Preparation, Amplification, Western Blot

    20) Product Images from "Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation"

    Article Title: Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation

    Journal: eLife

    doi: 10.7554/eLife.02758

    Appropriate ParB-stimulated ParA ATPase rates are important for the robustness of the DNA-relay model. ( A ) Averaged trajectories of the partition complex along the long cell axis during the fast ParA-dependent phase were simulated using varied ParB-stimulated ParA ATPase rates ( k cat ) and a fixed diffusion coefficient for the ParB/ parS complex ( D PC ) of 0.0001 μm 2 /s. ( B ) Same data set as ( A ), except that for each k cat , the fraction of trajectories that completed translocation are shown as a function of time. DOI: http://dx.doi.org/10.7554/eLife.02758.025
    Figure Legend Snippet: Appropriate ParB-stimulated ParA ATPase rates are important for the robustness of the DNA-relay model. ( A ) Averaged trajectories of the partition complex along the long cell axis during the fast ParA-dependent phase were simulated using varied ParB-stimulated ParA ATPase rates ( k cat ) and a fixed diffusion coefficient for the ParB/ parS complex ( D PC ) of 0.0001 μm 2 /s. ( B ) Same data set as ( A ), except that for each k cat , the fraction of trajectories that completed translocation are shown as a function of time. DOI: http://dx.doi.org/10.7554/eLife.02758.025

    Techniques Used: Diffusion-based Assay, Translocation Assay

    L12A mutation, which prevents ParA–ParB interaction, severely compromises ParB ability to activate ParA ATPase activity. ATPase rates were measured from reactions containing 3 µM ParA and 1.5 mg/ml salmon sperm DNA in the absence or presence of 90 µM of either wild-type ParB or ParB L12A . The measured rates were compared to the rate of the reaction lacking ParB. Error bars represent the standard deviations (SD) of results measured in duplicates. DOI: http://dx.doi.org/10.7554/eLife.02758.005
    Figure Legend Snippet: L12A mutation, which prevents ParA–ParB interaction, severely compromises ParB ability to activate ParA ATPase activity. ATPase rates were measured from reactions containing 3 µM ParA and 1.5 mg/ml salmon sperm DNA in the absence or presence of 90 µM of either wild-type ParB or ParB L12A . The measured rates were compared to the rate of the reaction lacking ParB. Error bars represent the standard deviations (SD) of results measured in duplicates. DOI: http://dx.doi.org/10.7554/eLife.02758.005

    Techniques Used: Mutagenesis, Activity Assay

    21) Product Images from "Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor"

    Article Title: Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor

    Journal: eLife

    doi: 10.7554/eLife.30688

    Mutagenesis used to identify the residues in the DBD that mediate the coupling to the R-domain. ( a ) Sequence conservation analysis of the DBD among the GRs in different species and among all members in the steroid hormone receptor family. Above is the secondary structure annotation of the GR DBD. Red lines label mutations on DBD investigated in this study. ( b ) Influence on the transcriptional activity of single point mutations carried out within DBD, on the C3 isoform. Mutations (L422A, S425G, C431Y, V435A, L436A, L482Y and Q483E) did not influence DNA-binding affinity or the coupling between DBD and F-domain, as these mutations did not significantly decrease the transcriptional activity. ( c ) Competitive transfection assay testing mutations (L422A, S425G, C431Y, V435A, L436A, V449A, L482Y and T493L) identified in Panel b using the linker-DBD and R A -linker-DBD constructs. Curves are shown for the mutations with different effect on the two constructs. ( d ) EC 50 was fitted from each competitive transfection curve (shown in c) and expressed as relative binding affinity to wild-type construct. ( e ) Luciferase assay dosage curves for the triple mutants A C431Y V435A L436A and C3 C431Y V435A L436A compared to their respective wild types A and C3. Curves are fitted to the data using the dose-response function, F ( C ) = 1 + A max − 1 1 + ( E C 50 / C ) P . Data fitting details are described in the Supplementary Figure 1a legend. ( f ) Maximum transcriptional activity and binding affinity of the triple mutants of A and C3 isoforms expressed as relative to their wild types. The effect on transcriptional activity and binding affinity of A and C3 isoforms by mutating residues on DBD can be predicted by the EAM for three scenarios: decreasing the DBD stability ( g ), decreasing the coupling between the F-domain and the DBD ( h ), and decreasing the coupling between the R-domain and the DBD (i). Comparing the experimental result (shown in panel f) with the EAM predictions confirms that the triple mutations C431Y/V435A/L436A significantly decrease the coupling between the R-domain and the DBD.
    Figure Legend Snippet: Mutagenesis used to identify the residues in the DBD that mediate the coupling to the R-domain. ( a ) Sequence conservation analysis of the DBD among the GRs in different species and among all members in the steroid hormone receptor family. Above is the secondary structure annotation of the GR DBD. Red lines label mutations on DBD investigated in this study. ( b ) Influence on the transcriptional activity of single point mutations carried out within DBD, on the C3 isoform. Mutations (L422A, S425G, C431Y, V435A, L436A, L482Y and Q483E) did not influence DNA-binding affinity or the coupling between DBD and F-domain, as these mutations did not significantly decrease the transcriptional activity. ( c ) Competitive transfection assay testing mutations (L422A, S425G, C431Y, V435A, L436A, V449A, L482Y and T493L) identified in Panel b using the linker-DBD and R A -linker-DBD constructs. Curves are shown for the mutations with different effect on the two constructs. ( d ) EC 50 was fitted from each competitive transfection curve (shown in c) and expressed as relative binding affinity to wild-type construct. ( e ) Luciferase assay dosage curves for the triple mutants A C431Y V435A L436A and C3 C431Y V435A L436A compared to their respective wild types A and C3. Curves are fitted to the data using the dose-response function, F ( C ) = 1 + A max − 1 1 + ( E C 50 / C ) P . Data fitting details are described in the Supplementary Figure 1a legend. ( f ) Maximum transcriptional activity and binding affinity of the triple mutants of A and C3 isoforms expressed as relative to their wild types. The effect on transcriptional activity and binding affinity of A and C3 isoforms by mutating residues on DBD can be predicted by the EAM for three scenarios: decreasing the DBD stability ( g ), decreasing the coupling between the F-domain and the DBD ( h ), and decreasing the coupling between the R-domain and the DBD (i). Comparing the experimental result (shown in panel f) with the EAM predictions confirms that the triple mutations C431Y/V435A/L436A significantly decrease the coupling between the R-domain and the DBD.

    Techniques Used: Mutagenesis, Sequencing, Activity Assay, Binding Assay, Transfection, Construct, Luciferase

    Transcriptional activity and DNA-binding affinity of GR translational isoforms. ( a ) Luciferase assay dosage curves for the constitutively active constructs of the eight GR translational isoforms. Per 30,000 cells, a constant 40 ng of GRE-driven luciferase vector was transfected, and the amount of GR vector co-transfected was increased from 0 ng to 5 ng. Errors are calculated from three samples. Curves are fitted to the data using the dose-response function, F ( C ) = 1 + A max − 1 1 + ( E C 50 / C ) P . A max represents the maximum transcriptional activity for each construct, and EC 50 represents the amount of GR construct transfected at the half-maximum transcriptional activity. C is the amount of GR construct transfected at each data point. p is an empirical value introduced in the fitting equation, to transform DNA vector amount to protein expression amount, to account for the possibility that different isoforms have different degradation rates, expression levels, nuclear localizations and/or cooperativities. Inset: Western blot showing that U-2 OS cells transiently transfected with the expression plasmid for each isoform express each of them. ( b ) Luciferase assay dosage curve keeping each GR isoform vector constant at 4 ng per 30,000 cells, while gradually increasing the GRE-driven luciferase vector from 0 ng to 40 ng. Data were fitted by a linear function. (This indicates that 5 ng GR isoform construct can saturate the 40 ng GRE driven luciferase vector.) Errors are the standard deviations of three independent samples. ( c ) Fluorescence anisotropy of the 6-FAM-labeled half site GRE (5’-gcgcAGAACAggagcgc-3’) as a function of GR translational isoform concentration. Binding was conducted with 25 nM 6-FAM labeled GRE in buffer containing 10 mM HEPES (pH7.6), 80 mM NaCl, 1 mM EDTA, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol, 200 ug/mL BSA and 5 µM control non-specific 17-mer oligo. Curves represent fits to the data with a single-site-binding model. ( d ) Correlation between the EC 50 fitted from the in vivo dosage curve as shown in panel a and the in vitro binding affinity as shown in panel c, demonstrating that the in vitro binding affinity represents the in vivo binding. Error bars represent uncertainty of the fits, as returned by the default settings of Mathematica’s NonLinearModelFit function. ( e ) Competitive transfection assay comparing D1, D2, D3 and DBD, against titration of a constant amount of C3 isoform (which has the highest activity). The transcription activity of C3 isoform in absence of competitors was normalized to 1. Data were fitted with dose-response function, F ( C ) = 1 + A c o m p e t i t o r − 1 1 + ( E C 50 / C ) P . In this equation, A competitor represents the transcriptional activity when 16 ng of competitor is transfected alone, EC 50 represents the co-transfected amount of competitor construct that results in half the activity of the C3 maximum. C is the amount of competitor construct transfected at each data point. p is an empirical value described in panel a. Inset: correlation between the EC 50 fitted from the competitive binding assay and the in vitro measured binding affinity for D1, D2 and D3 isoforms (as obtained from panel c). Errors reflect uncertainties of the individual fits, as returned by the default settings of Mathematica’s NonLinearModelFit function. The correlation demonstrates that the competitive transfection assay provides qualitative information about the binding affinity of each inactive construct. ( f ). Multicolor immunostaining of U-2 OS cells transfected with A, B, C1, C2, C3, D1, D2 and D3 constructs. Green: Alexa 488 linked goat anti-mouse IgG staining GR. Blue: DAPI staining nuclei. Red: Rhodamine Phalloidin staining F-actin. ( g ) Nuclear localization efficiency for the eight GR translational isoforms. Nuclear percentage is calculated by dividing the intensity of the green dye overlapped with blue dye with the total green dye intensity as shown in panel f. Three pictures were used for each isoform for the quantification. Average values and standard errors of the mean are reported in the graph.
    Figure Legend Snippet: Transcriptional activity and DNA-binding affinity of GR translational isoforms. ( a ) Luciferase assay dosage curves for the constitutively active constructs of the eight GR translational isoforms. Per 30,000 cells, a constant 40 ng of GRE-driven luciferase vector was transfected, and the amount of GR vector co-transfected was increased from 0 ng to 5 ng. Errors are calculated from three samples. Curves are fitted to the data using the dose-response function, F ( C ) = 1 + A max − 1 1 + ( E C 50 / C ) P . A max represents the maximum transcriptional activity for each construct, and EC 50 represents the amount of GR construct transfected at the half-maximum transcriptional activity. C is the amount of GR construct transfected at each data point. p is an empirical value introduced in the fitting equation, to transform DNA vector amount to protein expression amount, to account for the possibility that different isoforms have different degradation rates, expression levels, nuclear localizations and/or cooperativities. Inset: Western blot showing that U-2 OS cells transiently transfected with the expression plasmid for each isoform express each of them. ( b ) Luciferase assay dosage curve keeping each GR isoform vector constant at 4 ng per 30,000 cells, while gradually increasing the GRE-driven luciferase vector from 0 ng to 40 ng. Data were fitted by a linear function. (This indicates that 5 ng GR isoform construct can saturate the 40 ng GRE driven luciferase vector.) Errors are the standard deviations of three independent samples. ( c ) Fluorescence anisotropy of the 6-FAM-labeled half site GRE (5’-gcgcAGAACAggagcgc-3’) as a function of GR translational isoform concentration. Binding was conducted with 25 nM 6-FAM labeled GRE in buffer containing 10 mM HEPES (pH7.6), 80 mM NaCl, 1 mM EDTA, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol, 200 ug/mL BSA and 5 µM control non-specific 17-mer oligo. Curves represent fits to the data with a single-site-binding model. ( d ) Correlation between the EC 50 fitted from the in vivo dosage curve as shown in panel a and the in vitro binding affinity as shown in panel c, demonstrating that the in vitro binding affinity represents the in vivo binding. Error bars represent uncertainty of the fits, as returned by the default settings of Mathematica’s NonLinearModelFit function. ( e ) Competitive transfection assay comparing D1, D2, D3 and DBD, against titration of a constant amount of C3 isoform (which has the highest activity). The transcription activity of C3 isoform in absence of competitors was normalized to 1. Data were fitted with dose-response function, F ( C ) = 1 + A c o m p e t i t o r − 1 1 + ( E C 50 / C ) P . In this equation, A competitor represents the transcriptional activity when 16 ng of competitor is transfected alone, EC 50 represents the co-transfected amount of competitor construct that results in half the activity of the C3 maximum. C is the amount of competitor construct transfected at each data point. p is an empirical value described in panel a. Inset: correlation between the EC 50 fitted from the competitive binding assay and the in vitro measured binding affinity for D1, D2 and D3 isoforms (as obtained from panel c). Errors reflect uncertainties of the individual fits, as returned by the default settings of Mathematica’s NonLinearModelFit function. The correlation demonstrates that the competitive transfection assay provides qualitative information about the binding affinity of each inactive construct. ( f ). Multicolor immunostaining of U-2 OS cells transfected with A, B, C1, C2, C3, D1, D2 and D3 constructs. Green: Alexa 488 linked goat anti-mouse IgG staining GR. Blue: DAPI staining nuclei. Red: Rhodamine Phalloidin staining F-actin. ( g ) Nuclear localization efficiency for the eight GR translational isoforms. Nuclear percentage is calculated by dividing the intensity of the green dye overlapped with blue dye with the total green dye intensity as shown in panel f. Three pictures were used for each isoform for the quantification. Average values and standard errors of the mean are reported in the graph.

    Techniques Used: Activity Assay, Binding Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Expressing, Western Blot, Fluorescence, Labeling, Concentration Assay, In Vivo, In Vitro, Titration, Competitive Binding Assay, Immunostaining, Staining

    22) Product Images from "Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress"

    Article Title: Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks849

    PIKK phosphorylation of RPA32. ( A–C ) In vitro phosphorylation of RPA32 with purified CDK, PIKKs or kinase-dead ATR, as indicated above each lane. Total RPA32 and phospho-specific forms were detected with indicated antibodies by western blot. Phosphorylated forms migrate slower (indicated by ‘p’). In panels A and C, control reactions lacking ATP are shown. ( D ) UM-SCC-38 cells expressing WT or S4A/S8A RPA32 were treated for 2 h with 100 µM etoposide, and with 10 µM KU55933 (ATMi), 20 µM NU7026 (DNA-PKi) 1 h prior to etoposide and added back after etoposide removal or mock treated as indicated, whole cell extracts were prepared and RPA32, and specific phospho-forms, were detected by western blot using indicated antibodies.
    Figure Legend Snippet: PIKK phosphorylation of RPA32. ( A–C ) In vitro phosphorylation of RPA32 with purified CDK, PIKKs or kinase-dead ATR, as indicated above each lane. Total RPA32 and phospho-specific forms were detected with indicated antibodies by western blot. Phosphorylated forms migrate slower (indicated by ‘p’). In panels A and C, control reactions lacking ATP are shown. ( D ) UM-SCC-38 cells expressing WT or S4A/S8A RPA32 were treated for 2 h with 100 µM etoposide, and with 10 µM KU55933 (ATMi), 20 µM NU7026 (DNA-PKi) 1 h prior to etoposide and added back after etoposide removal or mock treated as indicated, whole cell extracts were prepared and RPA32, and specific phospho-forms, were detected by western blot using indicated antibodies.

    Techniques Used: In Vitro, Purification, Western Blot, Expressing

    23) Product Images from "RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair"

    Article Title: RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair

    Journal: Nature Communications

    doi: 10.1038/ncomms10529

    Aberrant V(D)J recombination leads to genomic instability in Rag2 c/c XLF −/− p53 −/− B cells. ( a – c ) Genomic instability at the Igk locus in v-abl pro-B cell lines. ( a ) Schematic representation of the Igk locus, with positions of the BACs used for generation of DNA FISH probes indicated. ( b ) Representative metaphase from Rag2 c/c XLF −/− p53 −/ − v-abl pro-B cell lines using the Igk C BAC probe (red) combined with Igk V BAC probe (green) and chromosome 6 paint (white). Yellow arrowheads point to broken or translocated chromosome 6. ( c ) Percentage of aberrant metaphases from v-abl pro-B cell lines of the indicated genotype harbouring chromosomes breaks (white) or translocations (black) involving the Igk locus. Histograms represent means+s.e.m. of two to three independent cell lines (see also Supplementary Table 3 ). *** P
    Figure Legend Snippet: Aberrant V(D)J recombination leads to genomic instability in Rag2 c/c XLF −/− p53 −/− B cells. ( a – c ) Genomic instability at the Igk locus in v-abl pro-B cell lines. ( a ) Schematic representation of the Igk locus, with positions of the BACs used for generation of DNA FISH probes indicated. ( b ) Representative metaphase from Rag2 c/c XLF −/− p53 −/ − v-abl pro-B cell lines using the Igk C BAC probe (red) combined with Igk V BAC probe (green) and chromosome 6 paint (white). Yellow arrowheads point to broken or translocated chromosome 6. ( c ) Percentage of aberrant metaphases from v-abl pro-B cell lines of the indicated genotype harbouring chromosomes breaks (white) or translocations (black) involving the Igk locus. Histograms represent means+s.e.m. of two to three independent cell lines (see also Supplementary Table 3 ). *** P

    Techniques Used: Fluorescence In Situ Hybridization, BAC Assay

    24) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    25) Product Images from "The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae"

    Article Title: The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01944-14

    The T2S system does not secrete the DNase Dns. DNA zymography of culture supernatants from the WT V. cholerae strain N16961 and V. cholerae Δ epsD was performed. DNase activity is detected by the clearing of DNA from the SDS-PAGE gel. As controls,
    Figure Legend Snippet: The T2S system does not secrete the DNase Dns. DNA zymography of culture supernatants from the WT V. cholerae strain N16961 and V. cholerae Δ epsD was performed. DNase activity is detected by the clearing of DNA from the SDS-PAGE gel. As controls,

    Techniques Used: Zymography, Activity Assay, SDS Page

    26) Product Images from "Effect of Bovine Papillomavirus E2 Protein-Specific Monoclonal Antibodies on Papillomavirus DNA Replication"

    Article Title: Effect of Bovine Papillomavirus E2 Protein-Specific Monoclonal Antibodies on Papillomavirus DNA Replication

    Journal: Journal of Virology

    doi:

    Characterization of E2-specific MAbs. (A) Reactivity of E2-specific MAbs with the native E2 protein. The mobility shift assay was carried out with 2 ng of bacterially expressed and purified E2 protein and 0.2 ng of radiolabelled E2 binding site for 15 min at room temperature. (B) Lanes 1 to 10 show reactivity of MAbs with discontinuous epitopes with truncated E2 proteins expressed in COS-7 cells. Band shift assays were performed with 2 μl of cell extract. Lanes 11 to 16 show reactivity of MAbs to the E2 DNA-binding domain (DBD). Bacterially expressed E2 protein was treated with 2 μg of pronase for 10 min at room temperature, and then reactivity was determined. (C) Reactivity of MAb 1E2 with truncated E2 proteins expressed in COS-7 cells. MAbs were added after E2 was mixed with its DNA target. neg., cells transfected with carrior only; wt, wild type. Protein-DNA complexes were resolved by 6% PAGE with 0.25× Tris-borate-EDTA.
    Figure Legend Snippet: Characterization of E2-specific MAbs. (A) Reactivity of E2-specific MAbs with the native E2 protein. The mobility shift assay was carried out with 2 ng of bacterially expressed and purified E2 protein and 0.2 ng of radiolabelled E2 binding site for 15 min at room temperature. (B) Lanes 1 to 10 show reactivity of MAbs with discontinuous epitopes with truncated E2 proteins expressed in COS-7 cells. Band shift assays were performed with 2 μl of cell extract. Lanes 11 to 16 show reactivity of MAbs to the E2 DNA-binding domain (DBD). Bacterially expressed E2 protein was treated with 2 μg of pronase for 10 min at room temperature, and then reactivity was determined. (C) Reactivity of MAb 1E2 with truncated E2 proteins expressed in COS-7 cells. MAbs were added after E2 was mixed with its DNA target. neg., cells transfected with carrior only; wt, wild type. Protein-DNA complexes were resolved by 6% PAGE with 0.25× Tris-borate-EDTA.

    Techniques Used: Mobility Shift, Purification, Binding Assay, Electrophoretic Mobility Shift Assay, Transfection, Polyacrylamide Gel Electrophoresis

    Effect of E2-specific MAbs on papillomavirus replication. (A) CHO4.15 cells constitutively expressing BPV-1 E1 and E2 proteins were electroporated with 100 ng of reporter plasmid pUCAlu and various concentrations of MAbs. Cells were harvested 72 h after electroporation. Episomal DNA was digested with Dpn I and linearizing enzyme Hin dIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a PhosphorImager, and signals from cells transfected with the origin-containing plasmid only were used as a control to normalize the results. Symbols: ●, MAb 3F12; ○, MAb 1E4; ▴, MAb 1H10; ■, MAb 5H4; □, nonspecific anti-β-galactosidase (β-gal) MAb. (B) Southern blot analysis of transient replication of the BPV-1 origin-containing plasmid pUCAlu in the CHO4.15 cell line in the presence of MAbs at a concentration of 80 μg/ml. Episomal DNA was extracted from cells 72 h after transfection. Filters were probed with radiolabelled plasmid pUCAlu. (C) Western blot analysis of E2 protein levels in transfected CHO4.15 cells with rabbit anti-E2 polyclonal antibody.
    Figure Legend Snippet: Effect of E2-specific MAbs on papillomavirus replication. (A) CHO4.15 cells constitutively expressing BPV-1 E1 and E2 proteins were electroporated with 100 ng of reporter plasmid pUCAlu and various concentrations of MAbs. Cells were harvested 72 h after electroporation. Episomal DNA was digested with Dpn I and linearizing enzyme Hin dIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a PhosphorImager, and signals from cells transfected with the origin-containing plasmid only were used as a control to normalize the results. Symbols: ●, MAb 3F12; ○, MAb 1E4; ▴, MAb 1H10; ■, MAb 5H4; □, nonspecific anti-β-galactosidase (β-gal) MAb. (B) Southern blot analysis of transient replication of the BPV-1 origin-containing plasmid pUCAlu in the CHO4.15 cell line in the presence of MAbs at a concentration of 80 μg/ml. Episomal DNA was extracted from cells 72 h after transfection. Filters were probed with radiolabelled plasmid pUCAlu. (C) Western blot analysis of E2 protein levels in transfected CHO4.15 cells with rabbit anti-E2 polyclonal antibody.

    Techniques Used: Expressing, Plasmid Preparation, Electroporation, Southern Blot, Transfection, Concentration Assay, Western Blot

    Effect of E2-specific Fab′ fragments on DNA replication. (A) Ability of MAb 5H4 and its Fab′ fragment to inhibit the formation of the E2-DNA complex at various antibody concentrations. The mobility shift assay was carried out with 2 ng of bacterially expressed and purified E2 protein and 0.2 ng of radiolabelled E2 binding site for 15 min at room temperature. MAb 5H4 or its Fab′ fragment was added after E2 was mixed with its DNA target, and incubation was carried out for an additional 20 min. (B) Inhibition of DNA replication by E2-specific Fab′ fragments at various concentrations. Reporter plasmid pUCAlu (100 ng) was cotransfected together with the Fab′ fragment of 3F12 (⧫), the Fab′ fragment of 1H10 (■), or the Fab′ fragment of 5H4 (▴) into cell line CHO4.15. At 72 h after electroporation, cells were harvested, and episomal DNA was digested with Dpn I and linearizing enzyme Hin dIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a PhosphorImager, and signals from cells transfected with the origin-containing plasmid only were used as a control to normalize the results. (C) Southern blot analysis of transient replication of the BPV-1 origin-containing plasmid pUCAlu in the CHO4.15 cell line in the presence of Fab′ fragments at a concentration of 0.3 mg/ml. Episomal DNA was extracted from cells either 48 or 72 h ( ) after transfection. Filters were probed with radiolabelled plasmid pUCAlu.
    Figure Legend Snippet: Effect of E2-specific Fab′ fragments on DNA replication. (A) Ability of MAb 5H4 and its Fab′ fragment to inhibit the formation of the E2-DNA complex at various antibody concentrations. The mobility shift assay was carried out with 2 ng of bacterially expressed and purified E2 protein and 0.2 ng of radiolabelled E2 binding site for 15 min at room temperature. MAb 5H4 or its Fab′ fragment was added after E2 was mixed with its DNA target, and incubation was carried out for an additional 20 min. (B) Inhibition of DNA replication by E2-specific Fab′ fragments at various concentrations. Reporter plasmid pUCAlu (100 ng) was cotransfected together with the Fab′ fragment of 3F12 (⧫), the Fab′ fragment of 1H10 (■), or the Fab′ fragment of 5H4 (▴) into cell line CHO4.15. At 72 h after electroporation, cells were harvested, and episomal DNA was digested with Dpn I and linearizing enzyme Hin dIII and analyzed by Southern blotting. The replication signals of three independent experiments were quantified with a PhosphorImager, and signals from cells transfected with the origin-containing plasmid only were used as a control to normalize the results. (C) Southern blot analysis of transient replication of the BPV-1 origin-containing plasmid pUCAlu in the CHO4.15 cell line in the presence of Fab′ fragments at a concentration of 0.3 mg/ml. Episomal DNA was extracted from cells either 48 or 72 h ( ) after transfection. Filters were probed with radiolabelled plasmid pUCAlu.

    Techniques Used: Mobility Shift, Purification, Binding Assay, Incubation, Inhibition, Plasmid Preparation, Electroporation, Southern Blot, Transfection, Concentration Assay

    27) Product Images from "Small-Molecule Positive Allosteric Modulators of the β2"

    Article Title: Small-Molecule Positive Allosteric Modulators of the β2

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.111948

    Hit compounds from DEL screening with the agonist-occupied β 2 AR in HDL particles. (A) Cartoon for DEL screening. Purified human β 2 ARs were reconstituted in HDL particles ( β 2 AR Nanodiscs) and then occupied by BI-167107 (BI). DNA-encoded library molecules were mixed with the BI-occupied β 2 AR Nanodiscs immobilized on NeutrAvidin beads through biotin–avidin interaction of biotinylated membrane scaffolding protein ApoA1. Three rounds of iterative selection were performed with each library. (B) Structures of the Cmpd-6 and six other primary hits. These compounds have varied chemical scaffolds in a common region, designated as R1. The different chemical structures in the R1 region of each analog are illustrated. (C) Analysis of Cmpd-6 for its physical interaction with the agonist-bound, active β 2 AR by ITC. The thermogram (insert) and binding isotherm with the best titration curve fit are shown. One site model was used to fit the data. Data are representative of three independent experiments. The values summarizing binding affinity (K D ), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) are shown in box below the graph.
    Figure Legend Snippet: Hit compounds from DEL screening with the agonist-occupied β 2 AR in HDL particles. (A) Cartoon for DEL screening. Purified human β 2 ARs were reconstituted in HDL particles ( β 2 AR Nanodiscs) and then occupied by BI-167107 (BI). DNA-encoded library molecules were mixed with the BI-occupied β 2 AR Nanodiscs immobilized on NeutrAvidin beads through biotin–avidin interaction of biotinylated membrane scaffolding protein ApoA1. Three rounds of iterative selection were performed with each library. (B) Structures of the Cmpd-6 and six other primary hits. These compounds have varied chemical scaffolds in a common region, designated as R1. The different chemical structures in the R1 region of each analog are illustrated. (C) Analysis of Cmpd-6 for its physical interaction with the agonist-bound, active β 2 AR by ITC. The thermogram (insert) and binding isotherm with the best titration curve fit are shown. One site model was used to fit the data. Data are representative of three independent experiments. The values summarizing binding affinity (K D ), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) are shown in box below the graph.

    Techniques Used: Purification, Avidin-Biotin Assay, Scaffolding, Selection, Binding Assay, Titration

    28) Product Images from "Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling"

    Article Title: Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2018.10.007

    N-Terminal Ub Promotes TRIM5 Immune Signaling, Chain Anchoring, and Proteasomal Degradation (A) Representative confocal fluorescence images of 293T transfected with TRIM5-HA or Ub(GA)TRIM5-HA, NF-κB-Luc, and RLuc, detecting HA tag or DNA, and frequency of CB-containing cells quantified. (B) Top: NF-κB-Luc activation in cells from (A). Bottom: immunoblot detecting HA tag and β-actin. (C) 2N/V2∼Ub discharge experiments with Ube2N(K92R). Left: reactions assessed by immunoblot detecting 2N (top) or TRIM5 (bottom two panels). Asterisk indicates contaminant protein. Right: densitometry of immunoblot shown. (D) LC-MS/MS analysis of 293T-expressed Ub(GA)TRIM5-His identifies four sites of Ub conjugation in N-monoUb, as inferred by modification of Lys with diGly (GG). Ubiquitinated peptides shown in blue. Peptide confidence in parentheses. (E) Immunoblot of in vitro reactions between TRIM5 RB, Ub(GA)RB, or Ub(K63R,GA)RB and 2N/V2, detecting TRIM5 (top) or Ub (bottom). (F and G) Top: NF-κB-FLuc activation in 293T by TRIM5 (F) or GFP (G) constructs indicated. Bottom: immunoblot detecting HA tag and β-actin. (H) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB or Ub(GA)RB(E11R) and 2N/V2, detecting TRIM5. (I) Top: NF-κB-Luc activation in 293T by constructs indicated. Bottom: immunoblot detecting the HA tag and β-actin. (J) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB, first conjugated with N-K63-Ub by 2N/V2, and proteasomes, incubated for 1–15 hr, detecting TRIM5. (K) Left: Coomassie-stained gel of reactions between Ub(GA)RB with and without prior Ub conjugation by 2N/V2, and proteasomes. Bands excised for LC-MS/MS analysis labeled a–d. X indicates the degradation product. Right: unique Ub(GA)RB peptides detected. Bottom: regions (i–iii) of Ub(GA)RB from which specific peptides were detected. Representative of three experiments. (L) Model, TRIM5 stimulates NF-κB and proteasome degradation via an N-K63-Ub tag. All data are representative of at least two replicates. Error bars represent SD. See also Figure S5 .
    Figure Legend Snippet: N-Terminal Ub Promotes TRIM5 Immune Signaling, Chain Anchoring, and Proteasomal Degradation (A) Representative confocal fluorescence images of 293T transfected with TRIM5-HA or Ub(GA)TRIM5-HA, NF-κB-Luc, and RLuc, detecting HA tag or DNA, and frequency of CB-containing cells quantified. (B) Top: NF-κB-Luc activation in cells from (A). Bottom: immunoblot detecting HA tag and β-actin. (C) 2N/V2∼Ub discharge experiments with Ube2N(K92R). Left: reactions assessed by immunoblot detecting 2N (top) or TRIM5 (bottom two panels). Asterisk indicates contaminant protein. Right: densitometry of immunoblot shown. (D) LC-MS/MS analysis of 293T-expressed Ub(GA)TRIM5-His identifies four sites of Ub conjugation in N-monoUb, as inferred by modification of Lys with diGly (GG). Ubiquitinated peptides shown in blue. Peptide confidence in parentheses. (E) Immunoblot of in vitro reactions between TRIM5 RB, Ub(GA)RB, or Ub(K63R,GA)RB and 2N/V2, detecting TRIM5 (top) or Ub (bottom). (F and G) Top: NF-κB-FLuc activation in 293T by TRIM5 (F) or GFP (G) constructs indicated. Bottom: immunoblot detecting HA tag and β-actin. (H) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB or Ub(GA)RB(E11R) and 2N/V2, detecting TRIM5. (I) Top: NF-κB-Luc activation in 293T by constructs indicated. Bottom: immunoblot detecting the HA tag and β-actin. (J) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB, first conjugated with N-K63-Ub by 2N/V2, and proteasomes, incubated for 1–15 hr, detecting TRIM5. (K) Left: Coomassie-stained gel of reactions between Ub(GA)RB with and without prior Ub conjugation by 2N/V2, and proteasomes. Bands excised for LC-MS/MS analysis labeled a–d. X indicates the degradation product. Right: unique Ub(GA)RB peptides detected. Bottom: regions (i–iii) of Ub(GA)RB from which specific peptides were detected. Representative of three experiments. (L) Model, TRIM5 stimulates NF-κB and proteasome degradation via an N-K63-Ub tag. All data are representative of at least two replicates. Error bars represent SD. See also Figure S5 .

    Techniques Used: Fluorescence, Transfection, Activation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Conjugation Assay, Modification, In Vitro, Construct, Incubation, Staining, Labeling

    Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.
    Figure Legend Snippet: Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.

    Techniques Used: Expressing, Western Blot, Transduction, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    TRIM5 Cytoplasmic Bodies Are Signaling Platforms (A and B) (A) Differentiated THP-1 cells infected with N- or B-MLV vector, or media alone, and mRNA fold inductions relative to media were quantified (ΔΔCt) by qPCR using ACTB as an uninduced control. In (B), THP-1 expressed control (shScr) or TRIM5-specific (shTRIM5) small hairpin RNA (shRNA). (C) THP-1 from (B) were infected with N- or B-MLV-GFP and percentage infection quantified by flow cytometry. (D) Differentiated THP-1 cells infected with N- or B-MLV, or 10 ng/mL TNFα, for times indicated. Immunoblots detecting phospho-IκBα, IκBα, and β-actin. (E) Representative confocal fluorescence images of differentiated THP-1 cells, infected with N- or B-MLV for the times indicated, detecting p65 or DNA. (F and G) TRIM5 , PTGS2 (F), and IL6 (G) expression relative to ACTB in 293T following transfection with WT or E11R (G) TRIM5. (H) NF-κB-Luc activation in 293T by WT or R119E TRIM5. (I) Immunoblot of WT or R119E TRIM5-His pull-downs (PDs) from 293T cells, detecting Ub in PD fraction and TRIM5 or β-actin in input fraction. (J) Representative confocal fluorescence images of 293T transfected with increasing doses of TRIM5 plasmid (bearing C-terminal HA tag), NF-κB-FLuc, and RLuc, detecting HA tag or DNA. Cells classified as containing diffuse TRIM5, CBs, aggregated TRIM5 (aggregates), or aggregates and CBs. Cells counted in eight fields of view, and different cell classes represented as parts of a whole. (K) NF-κB-FLuc activation in cells from (J), plotted against the number of cells at each plasmid dose containing either CB or aggregates. (L) NF-κB-Luc activation in 293T by WT, E11R, L19R, or I76R TRIM5. All data are representative of at least two replicates. All data are mean values; error bars represent SD. See also Figure S3 .
    Figure Legend Snippet: TRIM5 Cytoplasmic Bodies Are Signaling Platforms (A and B) (A) Differentiated THP-1 cells infected with N- or B-MLV vector, or media alone, and mRNA fold inductions relative to media were quantified (ΔΔCt) by qPCR using ACTB as an uninduced control. In (B), THP-1 expressed control (shScr) or TRIM5-specific (shTRIM5) small hairpin RNA (shRNA). (C) THP-1 from (B) were infected with N- or B-MLV-GFP and percentage infection quantified by flow cytometry. (D) Differentiated THP-1 cells infected with N- or B-MLV, or 10 ng/mL TNFα, for times indicated. Immunoblots detecting phospho-IκBα, IκBα, and β-actin. (E) Representative confocal fluorescence images of differentiated THP-1 cells, infected with N- or B-MLV for the times indicated, detecting p65 or DNA. (F and G) TRIM5 , PTGS2 (F), and IL6 (G) expression relative to ACTB in 293T following transfection with WT or E11R (G) TRIM5. (H) NF-κB-Luc activation in 293T by WT or R119E TRIM5. (I) Immunoblot of WT or R119E TRIM5-His pull-downs (PDs) from 293T cells, detecting Ub in PD fraction and TRIM5 or β-actin in input fraction. (J) Representative confocal fluorescence images of 293T transfected with increasing doses of TRIM5 plasmid (bearing C-terminal HA tag), NF-κB-FLuc, and RLuc, detecting HA tag or DNA. Cells classified as containing diffuse TRIM5, CBs, aggregated TRIM5 (aggregates), or aggregates and CBs. Cells counted in eight fields of view, and different cell classes represented as parts of a whole. (K) NF-κB-FLuc activation in cells from (J), plotted against the number of cells at each plasmid dose containing either CB or aggregates. (L) NF-κB-Luc activation in 293T by WT, E11R, L19R, or I76R TRIM5. All data are representative of at least two replicates. All data are mean values; error bars represent SD. See also Figure S3 .

    Techniques Used: Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, shRNA, Flow Cytometry, Cytometry, Western Blot, Fluorescence, Expressing, Transfection, Activation Assay

    Ubiquitination Is Redundant for Overall Viral Restriction (A) B-MLV reverse transcribes its genome and integrates into the host. N-MLV recruits TRIM5, is disassembled, and cannot reverse transcribe. TRIM5 stimulates intracellular signaling and transcriptional upregulation of antiviral genes. The timing of Ub during restriction is contested. (B) Structure of the rhesus macaque TRIM5 RING dimer in complex with Ube2N (2N) (PDB: 4TKP ). (C and D) In vitro ubiquitination experiments between TRIM5 RING-B-Box (RB) mutants and Ube2W (C), Ube2N/Ube2V2 (2N/V2) (C and D) or both Ube2W and 2N/V2 (C and D), detecting TRIM5 or Ub. (E and F) CrFK cells expressing vector or full-length TRIM5 mutants (bearing C-terminal hemagglutinin [HA] tags), infected with N-MLV- or B-MLV-GFP vectors. Percentage infection quantified by flow cytometry (E and F). Boiled virus served as a negative control (E). (G) Representative confocal fluorescence images of CrFK cells from (E) and (F), detecting HA tag or DNA. (H) Model. The RING domain determines the morphology and restriction potency, but not the formation, of TRIM5 assemblies. All experiments representative of at least triplicates. In (E) and (F), error bars represent SD; values are means ± SD.
    Figure Legend Snippet: Ubiquitination Is Redundant for Overall Viral Restriction (A) B-MLV reverse transcribes its genome and integrates into the host. N-MLV recruits TRIM5, is disassembled, and cannot reverse transcribe. TRIM5 stimulates intracellular signaling and transcriptional upregulation of antiviral genes. The timing of Ub during restriction is contested. (B) Structure of the rhesus macaque TRIM5 RING dimer in complex with Ube2N (2N) (PDB: 4TKP ). (C and D) In vitro ubiquitination experiments between TRIM5 RING-B-Box (RB) mutants and Ube2W (C), Ube2N/Ube2V2 (2N/V2) (C and D) or both Ube2W and 2N/V2 (C and D), detecting TRIM5 or Ub. (E and F) CrFK cells expressing vector or full-length TRIM5 mutants (bearing C-terminal hemagglutinin [HA] tags), infected with N-MLV- or B-MLV-GFP vectors. Percentage infection quantified by flow cytometry (E and F). Boiled virus served as a negative control (E). (G) Representative confocal fluorescence images of CrFK cells from (E) and (F), detecting HA tag or DNA. (H) Model. The RING domain determines the morphology and restriction potency, but not the formation, of TRIM5 assemblies. All experiments representative of at least triplicates. In (E) and (F), error bars represent SD; values are means ± SD.

    Techniques Used: In Vitro, Expressing, Plasmid Preparation, Infection, Flow Cytometry, Cytometry, Negative Control, Fluorescence

    N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.
    Figure Legend Snippet: N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Fluorescence, Construct, Real-time Polymerase Chain Reaction, Plasmid Preparation

    29) Product Images from "Phage TP901-1 Site-Specific Integrase Functions in Human Cells"

    Article Title: Phage TP901-1 Site-Specific Integrase Functions in Human Cells

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.184.13.3657-3663.2002

    Efficiency of integrative recombination mediated by TP901-1 integrase between att sites of various sizes in E. coli . For each sequence shown, the uppercase letters represent att DNA sequences, while the lowercase letters represent flanking vector sequences. Boldface type indicates the 5-bp common core, and the underlined sequence is the 7-bp identical region shared by attB and attP . Previously identified repeats are indicated by lines above the attB31 and attP50 sequences, as discussed in the text. The asterisks next to these two att sites indicate that they are the smallest sites that were still fully active in this assay. The recombination frequency is the intramolecular integration frequency, calculated by determining the ratio of white colonies to total colonies and multiplying by 100. Each att site is followed by a number indicating the length of the att DNA sequence. In attB30A and attB30B there were deletions from the left and right sides of the sequence, respectively. attP56ctr is an attP sequence in which the att base pairs are centered precisely around the 5-bp core. Each attB was tested by integrative recombination against a 333-bp attP , while each attP was tested against a 53-bp attB . Frequency calculations were made by using bacterial strain DH-TPInt and are based on total numbers of bacterial colonies ranging from 500 to 8,800.
    Figure Legend Snippet: Efficiency of integrative recombination mediated by TP901-1 integrase between att sites of various sizes in E. coli . For each sequence shown, the uppercase letters represent att DNA sequences, while the lowercase letters represent flanking vector sequences. Boldface type indicates the 5-bp common core, and the underlined sequence is the 7-bp identical region shared by attB and attP . Previously identified repeats are indicated by lines above the attB31 and attP50 sequences, as discussed in the text. The asterisks next to these two att sites indicate that they are the smallest sites that were still fully active in this assay. The recombination frequency is the intramolecular integration frequency, calculated by determining the ratio of white colonies to total colonies and multiplying by 100. Each att site is followed by a number indicating the length of the att DNA sequence. In attB30A and attB30B there were deletions from the left and right sides of the sequence, respectively. attP56ctr is an attP sequence in which the att base pairs are centered precisely around the 5-bp core. Each attB was tested by integrative recombination against a 333-bp attP , while each attP was tested against a 53-bp attB . Frequency calculations were made by using bacterial strain DH-TPInt and are based on total numbers of bacterial colonies ranging from 500 to 8,800.

    Techniques Used: Sequencing, Plasmid Preparation

    30) Product Images from "Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration"

    Article Title: Gene Capture Coupled to High-Throughput Sequencing as a Strategy for Targeted Metagenome Exploration

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst001

    Schematic comparison of targeted capture methods applied to classical direct selection method of individual genomic loci (human for instance) (A) and our new approach for metagenomics targeting (B). The enrichment through microarray and the SHS of large genomic regions within complex eukaryotic genomes, as described in A, uses specific tiling probes to target resequencing genomic loci for copy number variation (CNV) and single nucleotide polymorphism detection. Our SHS method (B) uses the design of specific variants and explorative probes across a targeted biomarker to specifically enrich large DNA fragments from complex metagenomic DNA. Captured DNA fragments are sequenced to explore biomarker diversity and adjacent flanking regions. The red rectangles indicate the targeted regions.
    Figure Legend Snippet: Schematic comparison of targeted capture methods applied to classical direct selection method of individual genomic loci (human for instance) (A) and our new approach for metagenomics targeting (B). The enrichment through microarray and the SHS of large genomic regions within complex eukaryotic genomes, as described in A, uses specific tiling probes to target resequencing genomic loci for copy number variation (CNV) and single nucleotide polymorphism detection. Our SHS method (B) uses the design of specific variants and explorative probes across a targeted biomarker to specifically enrich large DNA fragments from complex metagenomic DNA. Captured DNA fragments are sequenced to explore biomarker diversity and adjacent flanking regions. The red rectangles indicate the targeted regions.

    Techniques Used: Selection, Microarray, Biomarker Assay

    31) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.02177-17

    Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.
    Figure Legend Snippet: Association of K8 with ori-Lyt DNA is mediated by RNA. (A) Schematic illustration of the KSHV ori-Lyt core domain and DNA fragments that were used in the DNA affinity assay. (B) Three biotinylated ori-Lyt DNA fragments and an irrelevant DNA fragment from the ORF45 coding region as a control were prepared by PCR, conjugated on magnetic beads, and incubated with TPA-induced BCBL-1 nuclear extract with and without treatment with RNase A. After washing, samples were assayed by Western blotting with antibodies as indicated. (C) Binding of K8 to ori-Lyt DNA was determined in BAC16 (BAC WT) and BAC-K8GDDGR by ChIP assay with anti-K8 antibody. The positions of the amplicons (3.1F and 12F) are shown in panel A. The error bars indicate SD.

    Techniques Used: Polymerase Chain Reaction, Magnetic Beads, Incubation, Western Blot, Binding Assay, BAC Assay, Chromatin Immunoprecipitation

    T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.
    Figure Legend Snippet: T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.

    Techniques Used: Sonication, Incubation, Real-time Polymerase Chain Reaction, Purification, Quantitative RT-PCR, Amplification

    32) Product Images from "Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes"

    Article Title: Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23582-1

    Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.
    Figure Legend Snippet: Schematic illustration of in situ PLA using conventional and UnFold probes. ( a ) Conventional in situ PLA. ( b ) In situ PLA using UnFold probes. (i) After pairs of primary antibodies have bound a pair of interacting proteins (red and green) followed by washes, secondary conventional or UnFold in situ PLA probes are added, followed after an incubation by renewed washes. (ii) In the conventional design under ( a ) two more oligonucleotides are then added that can form a DNA circle. Using the UnFold design in ( b ) the probe carrying a hairpin-loop oligonucleotide is cleaved at the U residues, liberating a free 5′ end capable of being ligated to the 3′ end of the same DNA strand. Meanwhile, the U residues in the hairpin DNA strand of the other UnFold probe are cleaved presenting a single-stranded template for the enzymatic joining of the ends of the strand on the first UnFold probe. (iii) A DNA ligase is added to form DNA circles in the two variants of in situ PLA. (iv) Finally, phi29 DNA polymerase is added to initiate RCA primed by oligonucleotides on one of the antibodies, and fluorescent oligonucleotides are used to visualize the RCA products.

    Techniques Used: In Situ, Proximity Ligation Assay, Incubation

    33) Product Images from "KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome"

    Article Title: KSHV PAN RNA Associates with Demethylases UTX and JMJD3 to Activate Lytic Replication through a Physical Interaction with the Virus Genome

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002680

    Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.
    Figure Legend Snippet: Overexpression of K-Rta cannot complement BAC36CRΔPAN. (A) BACmid containing cell lines were transfected with a K-Rta expression plasmid and supernatant virus DNA was measured 4 days post transfection. The experiment was repeated 3 times. Error bars are the standard deviation from the mean. (B) Trans expression of K-Rta activates viral promoters in BAC36CRΔPAN containing cells and expression is enhanced in the presence of PAN RNA. BAC36CRΔPAN containing cells were transfected with K-Rta with or without the cotransfection of the PAN RNA expression plasmid and qPCR analysis was performed to measure mRNA accumulation for several viral encoded genes.

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Standard Deviation, Cotransfection, RNA Expression, Real-time Polymerase Chain Reaction

    JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.
    Figure Legend Snippet: JMJD3 and UTX demethylases interact with KSHV DNA in the presence of PAN RNA expression. Cell lines containing BAC36CR or BAC36CRΔPAN were transfected with a K-Rta expression plasmid and ChIP assays were performed 3 days post transfection. Immunoprecipitations were performed using antibodies specific for JMJD3, UTX, K-Rta or an isotype specific antibody control. PCR primers specific for the ORF50 promoter or ORF45 were used to amplify immunoprecipitated DNA. Panel BAC36CRΔPAN+PAN: cells were transfected with both a K-Rta and PAN RNA expression plasmid.

    Techniques Used: RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation

    PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.
    Figure Legend Snippet: PAN RNA interacts with demethylases and the histone methyltransferase MLL2. (A) TREx/BCBL-1 Rta cells were treated with DOX and RNA CLIP assays were performed 3 days post treatment. PAN RNA-protein complexes were immunoprecipitated using anti-JMJD3, anti-UTX, anti-MML2 or isotype control antibodies. PCR primers were used to amplify (after RT) PAN RNA, ORF45 RNA or U1 RNA. Also shown is PCR amplification without a reverse transcriptase reaction (PAN no RT). (B) PAN RNA expression leads to a relative decrease in the H3K27me3 mark on the ORF50 promoter. BAC36CR or BAC36CRΔPAN containing cells were transfected with either a K-Rta expression plasmid and/or a plasmid expressing PAN RNA. ChIP assays were performed using anti-H3K27me3 specific antibody. Immunoprecipitated DNA was analyzed by qPCR normalized to input DNA. Data is reported as fold decrease compared to BAC36CR untreated samples. Error bars are the standard deviation of the mean from three separate experiments.

    Techniques Used: Cross-linking Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification, RNA Expression, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation

    PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.
    Figure Legend Snippet: PAN RNA physically interacts with the ORF50 promoter. (A) PAN RNA is enriched 30-fold by ChIRP assay. PAN RNA or LacZ specific biotinylated oligonucleotides were used to enrich PAN RNA. Recovered RNA or RNA from the depleted lysate (post ChIRP) was measured by qPCR. (B) TREx/BCBL-1 Rta cells were treated with DOX and 3 days post treatment ChIRP assays were performed. Tiling biotinylated oligonucleotides were used that hybridized to either PAN RNA (20 oligonucleotides) or control LacZ RNA (20 oligonucleotides). Pulled down DNA that was occupied by RNA was amplified using primers specific for the ORF50 promoter region or K6 ORF coding sequence.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Sequencing

    Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.
    Figure Legend Snippet: Generation of a recombinant BACmid with the PAN RNA locus deleted, BAC36CRΔPAN. (A) The BAC36CR template was used to insert the GalK-KanR cassette such that 634 nts of the PAN RNA gene was removed from the genome. (B) The GalK-KanR cassette was removed by homologous recombination and reverse selection. (C) BAC36CRΔPAN was generated by removal of the GalK-KanR cassette and the putative polyadenlyation signal downstream of the original PAN RNA and K7 genes was preserved. (D) Ethidium bromide stained agarose gel and Southern blot of BAC36, BAC36CR and BAC36CRΔPAN DNA cleaved with BamHI showing the removal of part of the PAN RNA locus.

    Techniques Used: Recombinant, Homologous Recombination, Selection, Generated, Staining, Agarose Gel Electrophoresis, Southern Blot

    Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.
    Figure Legend Snippet: Deletion of the duplicated region within BAC36. (A) Duplicated genomic region is located between two terminal repeat sequences of the BAC36 genome (B) The duplicated ORFs 18-K5 were removed by insertion of a GalK-KanR cassette using oligonucleotides homologous to regions outside of the duplicated region. (C) Replacement of the GalK-KanR cassette in the BAC36 genome to yield the recombinant BACmid BAC36CR where the entire duplicated regions was removed. Shown is the DNA sequence after removal of the cassette (D) Ethidium bromide stained gel and Southern blot of BAC36 and BAC36CR DNA cleaved with BamHI and hybridized with either a probe specific for the GalK-KanR cassette (GalK probe) or the PAN RNA locus (PAN probe). Lanes: 1, MW marker; 2, BAC36, 3, BAC36+GalK-KanR cassette; 4, BAC36CR. Arrows indicated the PAN RNA locus in the unique long region of the genome and the duplicated PAN RNA locus located between the terminal repeats.

    Techniques Used: Recombinant, Sequencing, Staining, Southern Blot, Marker

    34) Product Images from "Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling"

    Article Title: Trivalent RING Assembly on Retroviral Capsids Activates TRIM5 Ubiquitination and Innate Immune Signaling

    Journal: Cell Host & Microbe

    doi: 10.1016/j.chom.2018.10.007

    N-Terminal Ub Promotes TRIM5 Immune Signaling, Chain Anchoring, and Proteasomal Degradation (A) Representative confocal fluorescence images of 293T transfected with TRIM5-HA or Ub(GA)TRIM5-HA, NF-κB-Luc, and RLuc, detecting HA tag or DNA, and frequency of CB-containing cells quantified. (B) Top: NF-κB-Luc activation in cells from (A). Bottom: immunoblot detecting HA tag and β-actin. (C) 2N/V2∼Ub discharge experiments with Ube2N(K92R). Left: reactions assessed by immunoblot detecting 2N (top) or TRIM5 (bottom two panels). Asterisk indicates contaminant protein. Right: densitometry of immunoblot shown. (D) LC-MS/MS analysis of 293T-expressed Ub(GA)TRIM5-His identifies four sites of Ub conjugation in N-monoUb, as inferred by modification of Lys with diGly (GG). Ubiquitinated peptides shown in blue. Peptide confidence in parentheses. (E) Immunoblot of in vitro reactions between TRIM5 RB, Ub(GA)RB, or Ub(K63R,GA)RB and 2N/V2, detecting TRIM5 (top) or Ub (bottom). (F and G) Top: NF-κB-FLuc activation in 293T by TRIM5 (F) or GFP (G) constructs indicated. Bottom: immunoblot detecting HA tag and β-actin. (H) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB or Ub(GA)RB(E11R) and 2N/V2, detecting TRIM5. (I) Top: NF-κB-Luc activation in 293T by constructs indicated. Bottom: immunoblot detecting the HA tag and β-actin. (J) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB, first conjugated with N-K63-Ub by 2N/V2, and proteasomes, incubated for 1–15 hr, detecting TRIM5. (K) Left: Coomassie-stained gel of reactions between Ub(GA)RB with and without prior Ub conjugation by 2N/V2, and proteasomes. Bands excised for LC-MS/MS analysis labeled a–d. X indicates the degradation product. Right: unique Ub(GA)RB peptides detected. Bottom: regions (i–iii) of Ub(GA)RB from which specific peptides were detected. Representative of three experiments. (L) Model, TRIM5 stimulates NF-κB and proteasome degradation via an N-K63-Ub tag. .
    Figure Legend Snippet: N-Terminal Ub Promotes TRIM5 Immune Signaling, Chain Anchoring, and Proteasomal Degradation (A) Representative confocal fluorescence images of 293T transfected with TRIM5-HA or Ub(GA)TRIM5-HA, NF-κB-Luc, and RLuc, detecting HA tag or DNA, and frequency of CB-containing cells quantified. (B) Top: NF-κB-Luc activation in cells from (A). Bottom: immunoblot detecting HA tag and β-actin. (C) 2N/V2∼Ub discharge experiments with Ube2N(K92R). Left: reactions assessed by immunoblot detecting 2N (top) or TRIM5 (bottom two panels). Asterisk indicates contaminant protein. Right: densitometry of immunoblot shown. (D) LC-MS/MS analysis of 293T-expressed Ub(GA)TRIM5-His identifies four sites of Ub conjugation in N-monoUb, as inferred by modification of Lys with diGly (GG). Ubiquitinated peptides shown in blue. Peptide confidence in parentheses. (E) Immunoblot of in vitro reactions between TRIM5 RB, Ub(GA)RB, or Ub(K63R,GA)RB and 2N/V2, detecting TRIM5 (top) or Ub (bottom). (F and G) Top: NF-κB-FLuc activation in 293T by TRIM5 (F) or GFP (G) constructs indicated. Bottom: immunoblot detecting HA tag and β-actin. (H) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB or Ub(GA)RB(E11R) and 2N/V2, detecting TRIM5. (I) Top: NF-κB-Luc activation in 293T by constructs indicated. Bottom: immunoblot detecting the HA tag and β-actin. (J) Immunoblot of in vitro reactions between TRIM5 Ub(GA)RB, first conjugated with N-K63-Ub by 2N/V2, and proteasomes, incubated for 1–15 hr, detecting TRIM5. (K) Left: Coomassie-stained gel of reactions between Ub(GA)RB with and without prior Ub conjugation by 2N/V2, and proteasomes. Bands excised for LC-MS/MS analysis labeled a–d. X indicates the degradation product. Right: unique Ub(GA)RB peptides detected. Bottom: regions (i–iii) of Ub(GA)RB from which specific peptides were detected. Representative of three experiments. (L) Model, TRIM5 stimulates NF-κB and proteasome degradation via an N-K63-Ub tag. .

    Techniques Used: Fluorescence, Transfection, Activation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Conjugation Assay, Modification, In Vitro, Construct, Incubation, Staining, Labeling

    Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.
    Figure Legend Snippet: Ubiquitination Drives TRIM5 Turnover and Virus Destruction (A and B) Left: cycloheximide (CHX) chase experiments of CrFK cells expressing full-length TRIM5 E2 interface mutants (A) or TRIM5 RING dimer mutants (B), bearing C-terminal HA tags (A and B). Immunoblots detecting HA tag or β-actin. Right: densitometry of immunoblots shown. (C and D) CrFK cells (C) or TE671 cells transduced with Cas9 and TRIM5-specific single guide RNA (D) and expressing empty vector or full-length TRIM5 mutants (bearing C-terminal HA tags), infected with N-MLV- or B-MLV-GFP vectors, viral DNA copies quantified by GFP TaqMan qPCR. In (C), vector or WT TRIM5 cells were also treated with 10 μM MG132. Boiled virus served as a control for plasmid contamination. (E) TE671 cells from (D) infected with N-MLV-GFP or B-MLV-GFP vectors, percentage infection quantified by flow cytometry. (F) Representative confocal fluorescence images of CrFK cells expressing full-length TRIM5 mutants (bearing C-terminal HA tags), detecting HA tag, K63-Ub, or DNA. Cells containing K63-Ub-positive CBs were counted and expressed as percentage of total cells counted. All data are representative of at least two replicates. (C–E) Error bars represent SD. In (F), n = number of cells counted in the experiment shown.

    Techniques Used: Expressing, Western Blot, Transduction, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    TRIM5 Cytoplasmic Bodies Are Signaling Platforms (A and B) (A) Differentiated THP-1 cells infected with N- or B-MLV vector, or media alone, and mRNA fold inductions relative to media were quantified (ΔΔCt) by qPCR using ACTB as an uninduced control. In (B), THP-1 expressed control (shScr) or TRIM5-specific (shTRIM5) small hairpin RNA (shRNA). (C) THP-1 from (B) were infected with N- or B-MLV-GFP and percentage infection quantified by flow cytometry. (D) Differentiated THP-1 cells infected with N- or B-MLV, or 10 ng/mL TNFα, for times indicated. Immunoblots detecting phospho-IκBα, IκBα, and β-actin. (E) Representative confocal fluorescence images of differentiated THP-1 cells, infected with N- or B-MLV for the times indicated, detecting p65 or DNA. (F and G) TRIM5 , PTGS2 (F), and IL6 (G) expression relative to ACTB in 293T following transfection with WT or E11R (G) TRIM5. (H) NF-κB-Luc activation in 293T by WT or R119E TRIM5. (I) Immunoblot of WT or R119E TRIM5-His pull-downs (PDs) from 293T cells, detecting Ub in PD fraction and TRIM5 or β-actin in input fraction. (J) Representative confocal fluorescence images of 293T transfected with increasing doses of TRIM5 plasmid (bearing C-terminal HA tag), NF-κB-FLuc, and RLuc, detecting HA tag or DNA. Cells classified as containing diffuse TRIM5, CBs, aggregated TRIM5 (aggregates), or aggregates and CBs. Cells counted in eight fields of view, and different cell classes represented as parts of a whole. (K) NF-κB-FLuc activation in cells from (J), plotted against the number of cells at each plasmid dose containing either CB or aggregates. (L) NF-κB-Luc activation in 293T by WT, E11R, L19R, or I76R TRIM5. .
    Figure Legend Snippet: TRIM5 Cytoplasmic Bodies Are Signaling Platforms (A and B) (A) Differentiated THP-1 cells infected with N- or B-MLV vector, or media alone, and mRNA fold inductions relative to media were quantified (ΔΔCt) by qPCR using ACTB as an uninduced control. In (B), THP-1 expressed control (shScr) or TRIM5-specific (shTRIM5) small hairpin RNA (shRNA). (C) THP-1 from (B) were infected with N- or B-MLV-GFP and percentage infection quantified by flow cytometry. (D) Differentiated THP-1 cells infected with N- or B-MLV, or 10 ng/mL TNFα, for times indicated. Immunoblots detecting phospho-IκBα, IκBα, and β-actin. (E) Representative confocal fluorescence images of differentiated THP-1 cells, infected with N- or B-MLV for the times indicated, detecting p65 or DNA. (F and G) TRIM5 , PTGS2 (F), and IL6 (G) expression relative to ACTB in 293T following transfection with WT or E11R (G) TRIM5. (H) NF-κB-Luc activation in 293T by WT or R119E TRIM5. (I) Immunoblot of WT or R119E TRIM5-His pull-downs (PDs) from 293T cells, detecting Ub in PD fraction and TRIM5 or β-actin in input fraction. (J) Representative confocal fluorescence images of 293T transfected with increasing doses of TRIM5 plasmid (bearing C-terminal HA tag), NF-κB-FLuc, and RLuc, detecting HA tag or DNA. Cells classified as containing diffuse TRIM5, CBs, aggregated TRIM5 (aggregates), or aggregates and CBs. Cells counted in eight fields of view, and different cell classes represented as parts of a whole. (K) NF-κB-FLuc activation in cells from (J), plotted against the number of cells at each plasmid dose containing either CB or aggregates. (L) NF-κB-Luc activation in 293T by WT, E11R, L19R, or I76R TRIM5. .

    Techniques Used: Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction, shRNA, Flow Cytometry, Cytometry, Western Blot, Fluorescence, Expressing, Transfection, Activation Assay

    Ubiquitination Is Redundant for Overall Viral Restriction (A) B-MLV reverse transcribes its genome and integrates into the host. N-MLV recruits TRIM5, is disassembled, and cannot reverse transcribe. TRIM5 stimulates intracellular signaling and transcriptional upregulation of antiviral genes. The timing of Ub during restriction is contested. ). (C and D) In vitro ubiquitination experiments between TRIM5 RING-B-Box (RB) mutants and Ube2W (C), Ube2N/Ube2V2 (2N/V2) (C and D) or both Ube2W and 2N/V2 (C and D), detecting TRIM5 or Ub. (E and F) CrFK cells expressing vector or full-length TRIM5 mutants (bearing C-terminal hemagglutinin [HA] tags), infected with N-MLV- or B-MLV-GFP vectors. Percentage infection quantified by flow cytometry (E and F). Boiled virus served as a negative control (E). (G) Representative confocal fluorescence images of CrFK cells from (E) and (F), detecting HA tag or DNA. (H) Model. The RING domain determines the morphology and restriction potency, but not the formation, of TRIM5 assemblies. All experiments representative of at least triplicates. In (E) and (F), error bars represent SD; values are means ± SD.
    Figure Legend Snippet: Ubiquitination Is Redundant for Overall Viral Restriction (A) B-MLV reverse transcribes its genome and integrates into the host. N-MLV recruits TRIM5, is disassembled, and cannot reverse transcribe. TRIM5 stimulates intracellular signaling and transcriptional upregulation of antiviral genes. The timing of Ub during restriction is contested. ). (C and D) In vitro ubiquitination experiments between TRIM5 RING-B-Box (RB) mutants and Ube2W (C), Ube2N/Ube2V2 (2N/V2) (C and D) or both Ube2W and 2N/V2 (C and D), detecting TRIM5 or Ub. (E and F) CrFK cells expressing vector or full-length TRIM5 mutants (bearing C-terminal hemagglutinin [HA] tags), infected with N-MLV- or B-MLV-GFP vectors. Percentage infection quantified by flow cytometry (E and F). Boiled virus served as a negative control (E). (G) Representative confocal fluorescence images of CrFK cells from (E) and (F), detecting HA tag or DNA. (H) Model. The RING domain determines the morphology and restriction potency, but not the formation, of TRIM5 assemblies. All experiments representative of at least triplicates. In (E) and (F), error bars represent SD; values are means ± SD.

    Techniques Used: In Vitro, Expressing, Plasmid Preparation, Infection, Flow Cytometry, Cytometry, Negative Control, Fluorescence

    N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.
    Figure Legend Snippet: N-Terminal Ubiquitination before TRIM5 Assembly Causes Premature Proteasome Recruitment (A and B) CrFK cells expressing WT or Ub(GA)TRIM5 RING mutants (A) or Ub mutants (B), infected with N-MLV- or B-MLV-GFP; percentage infection quantified by flow cytometry. (C) Representative confocal fluorescence images of CrFK cells expressing constructs indicated, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. (D) Number of visible CBs per cell, from (C). (E) CrFK cells from (C) infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry. (F and G) CrFK cells expressing constructs indicated, infected with N-MLV- or B-MLV-GFP, percentage infection quantified by flow cytometry (F) or viral DNA copies quantified by TaqMan GFP qPCR (G). In (G), cells were also treated with DMSO or 10 μM MG132. Boiled virus served as a control for plasmid contamination. (H) Representative confocal fluorescence images of CrFK cells expressing Ub(K63only,GA)TRIM5-HA, treated with DMSO or 10 μM MG132, detecting HA tag or DNA. All data are representative of at least two replicates. Error bars represent SD.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Fluorescence, Construct, Real-time Polymerase Chain Reaction, Plasmid Preparation

    35) Product Images from "Small-Molecule Positive Allosteric Modulators of the β2"

    Article Title: Small-Molecule Positive Allosteric Modulators of the β2

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.118.111948

    Hit compounds from DEL screening with the agonist-occupied β 2 AR in HDL particles. (A) Cartoon for DEL screening. Purified human β 2 ARs were reconstituted in HDL particles ( β 2 AR Nanodiscs) and then occupied by BI-167107 (BI). DNA-encoded library molecules were mixed with the BI-occupied β 2 AR Nanodiscs immobilized on NeutrAvidin beads through biotin–avidin interaction of biotinylated membrane scaffolding protein ApoA1. Three rounds of iterative selection were performed with each library. (B) Structures of the Cmpd-6 and six other primary hits. These compounds have varied chemical scaffolds in a common region, designated as R1. The different chemical structures in the R1 region of each analog are illustrated. (C) Analysis of Cmpd-6 for its physical interaction with the agonist-bound, active β 2 AR by ITC. The thermogram (insert) and binding isotherm with the best titration curve fit are shown. One site model was used to fit the data. Data are representative of three independent experiments. The values summarizing binding affinity (K D ), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) are shown in box below the graph.
    Figure Legend Snippet: Hit compounds from DEL screening with the agonist-occupied β 2 AR in HDL particles. (A) Cartoon for DEL screening. Purified human β 2 ARs were reconstituted in HDL particles ( β 2 AR Nanodiscs) and then occupied by BI-167107 (BI). DNA-encoded library molecules were mixed with the BI-occupied β 2 AR Nanodiscs immobilized on NeutrAvidin beads through biotin–avidin interaction of biotinylated membrane scaffolding protein ApoA1. Three rounds of iterative selection were performed with each library. (B) Structures of the Cmpd-6 and six other primary hits. These compounds have varied chemical scaffolds in a common region, designated as R1. The different chemical structures in the R1 region of each analog are illustrated. (C) Analysis of Cmpd-6 for its physical interaction with the agonist-bound, active β 2 AR by ITC. The thermogram (insert) and binding isotherm with the best titration curve fit are shown. One site model was used to fit the data. Data are representative of three independent experiments. The values summarizing binding affinity (K D ), stoichiometry (N), enthalpy (ΔH), and entropy (ΔS) are shown in box below the graph.

    Techniques Used: Purification, Avidin-Biotin Assay, Scaffolding, Selection, Binding Assay, Titration

    36) Product Images from "AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production"

    Article Title: AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

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

    doi: 10.1073/pnas.1702435114

    c-di-GMP–dependent binding of CuxR to a direct-repeat element located within the uxs1 promoter region. ( A ) CuxR specifically binds to c-di-GMP in DRaCALA. ( B ) CuxR requires c-di-GMP for binding to the intergenic region (IR) between cuxR and uxs1 in EMSA. ( C ) CuxR binds to a DNA fragment containing 196 bp upstream of the uxs1 start codon, but not to a fragment containing 186 bp, in the presence of c-di-GMP. ( D ) The IR between cuxR and uxs1 contains a direct repeat and a palindromic sequence motif. Mutations within both motifs are denoted as m1 to m6. ( E ) The IR from positions 166–195 upstream of the uxs1 start codon is sufficient for c-di-GMP–dependent CuxR interaction with the DNA in EMSA. ( F ) Mutations in the direct-repeat element but not within the palindrome of the IR abolish DNA binding by CuxR. ( G ) Overproduction of both CuxR and PleD in S. meliloti does not stimulate P uxs1 containing mutations in the direct-repeat element of the IR. Error bars indicate SDs of three biological replicates. RFU, relative EGFP fluorescence units.
    Figure Legend Snippet: c-di-GMP–dependent binding of CuxR to a direct-repeat element located within the uxs1 promoter region. ( A ) CuxR specifically binds to c-di-GMP in DRaCALA. ( B ) CuxR requires c-di-GMP for binding to the intergenic region (IR) between cuxR and uxs1 in EMSA. ( C ) CuxR binds to a DNA fragment containing 196 bp upstream of the uxs1 start codon, but not to a fragment containing 186 bp, in the presence of c-di-GMP. ( D ) The IR between cuxR and uxs1 contains a direct repeat and a palindromic sequence motif. Mutations within both motifs are denoted as m1 to m6. ( E ) The IR from positions 166–195 upstream of the uxs1 start codon is sufficient for c-di-GMP–dependent CuxR interaction with the DNA in EMSA. ( F ) Mutations in the direct-repeat element but not within the palindrome of the IR abolish DNA binding by CuxR. ( G ) Overproduction of both CuxR and PleD in S. meliloti does not stimulate P uxs1 containing mutations in the direct-repeat element of the IR. Error bars indicate SDs of three biological replicates. RFU, relative EGFP fluorescence units.

    Techniques Used: Binding Assay, Sequencing, Fluorescence

    37) Product Images from "Up-regulation of Ciliary Neurotrophic Factor in Astrocytes by Aspirin"

    Article Title: Up-regulation of Ciliary Neurotrophic Factor in Astrocytes by Aspirin

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.447268

    Aspirin induces the recruitment of CREB to the Cntf promoter. A, DNA sequence of the CNTF promoter region containing the CRE. Mouse astrocytes pretreated with 2 μ m H-89 for 30 min were stimulated with 10 μ m aspirin for 2 h under serum-free
    Figure Legend Snippet: Aspirin induces the recruitment of CREB to the Cntf promoter. A, DNA sequence of the CNTF promoter region containing the CRE. Mouse astrocytes pretreated with 2 μ m H-89 for 30 min were stimulated with 10 μ m aspirin for 2 h under serum-free

    Techniques Used: Sequencing

    38) Product Images from "A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti"

    Article Title: A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00801-18

    MucR counteracts c-di-GMP- and CuxR-mediated activation of the uxs1 promoter. (A) cuxR and uxs1 promoter activities in different S. meliloti genetic backgrounds measured using promoter- egfp fusions on medium-copy-number plasmid pSRKGm- egfp . (B) uxs1 promoter activity in different S. meliloti genetic backgrounds with (pABC- P syn -cuxR ) or without (empty vector pABC- P syn ) constitutively expressed cuxR . (A and B) c-di-GMP 0 ), was used as a background strain. Error bars represent the standard deviations from four biological replicates. (C) Interaction of His 6 -MucR with DNA containing the cuxR-uxs1 intergenic region ( cuxR-uxs1 IR ) or a 196-bp region upstream of uxs1 (P uxs1 ) assayed with EMSA. DNA containing either the upstream region of rpsB or that of mucR served as negative or positive control, respectively.
    Figure Legend Snippet: MucR counteracts c-di-GMP- and CuxR-mediated activation of the uxs1 promoter. (A) cuxR and uxs1 promoter activities in different S. meliloti genetic backgrounds measured using promoter- egfp fusions on medium-copy-number plasmid pSRKGm- egfp . (B) uxs1 promoter activity in different S. meliloti genetic backgrounds with (pABC- P syn -cuxR ) or without (empty vector pABC- P syn ) constitutively expressed cuxR . (A and B) c-di-GMP 0 ), was used as a background strain. Error bars represent the standard deviations from four biological replicates. (C) Interaction of His 6 -MucR with DNA containing the cuxR-uxs1 intergenic region ( cuxR-uxs1 IR ) or a 196-bp region upstream of uxs1 (P uxs1 ) assayed with EMSA. DNA containing either the upstream region of rpsB or that of mucR served as negative or positive control, respectively.

    Techniques Used: Activation Assay, Plasmid Preparation, Activity Assay, Positive Control

    39) Product Images from "Comparative RNA sequencing reveals that HPV16 E6 abrogates the effect of E6*I on ROS metabolism"

    Article Title: Comparative RNA sequencing reveals that HPV16 E6 abrogates the effect of E6*I on ROS metabolism

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42393-6

    Expression levels of E6- and E6*I-deregulated transcripts in W12 clones. ( A) RT-qPCR analysis showing E6 and E6*I absolute mRNA levels (copies per µL of pure cDNA) in W12_20861, W12_20862 and W12_20863 clones. pXJ40-E6 and pXJ40-E6*I diluted in salmon sperm DNA (50 ng/µL) were used to generate standard curves. (B) Western blot analysis showing HPV16 E6 and E7, p53 and pRb protein levels in W12 clones. β-actin is used as loading control. RT-qPCR analyses showing. Full-length gels are presented in Figure S7 . (C) SYCP2, TNFAIP6 and p21, and (D) CCL2, RAC2 and PDGFB relative mRNA expression in W12 clones. RPLP0 was used as housekeeping gene. RT-qPCR are represented as means +/− S.D. of 3 independent experiments.
    Figure Legend Snippet: Expression levels of E6- and E6*I-deregulated transcripts in W12 clones. ( A) RT-qPCR analysis showing E6 and E6*I absolute mRNA levels (copies per µL of pure cDNA) in W12_20861, W12_20862 and W12_20863 clones. pXJ40-E6 and pXJ40-E6*I diluted in salmon sperm DNA (50 ng/µL) were used to generate standard curves. (B) Western blot analysis showing HPV16 E6 and E7, p53 and pRb protein levels in W12 clones. β-actin is used as loading control. RT-qPCR analyses showing. Full-length gels are presented in Figure S7 . (C) SYCP2, TNFAIP6 and p21, and (D) CCL2, RAC2 and PDGFB relative mRNA expression in W12 clones. RPLP0 was used as housekeeping gene. RT-qPCR are represented as means +/− S.D. of 3 independent experiments.

    Techniques Used: Expressing, Clone Assay, Quantitative RT-PCR, Western Blot

    40) Product Images from "Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR"

    Article Title: Post-transcriptional regulation of Pabpn1 by the RNA binding protein HuR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky535

    The long Pabpn1 3′UTR contains putative conserved cis- regulatory elements. ( A ) Schematic of the Pabpn1 transcript including a 5′ untranslated region (5′UTR), the coding DNA sequence ( Pabpn1 CDS), which encodes the PABPN1 open reading frame, and a 3′ untranslated region (3′UTR) which contains two polyadenylation signals (PASI, PASII) and multiple putative AU-rich elements (ARE1, ARE2, ARE3, ARE4). ( B ) Schematic of qRT-PCR strategy using primers that recognize the coding DNA sequence (CDS primers) and distal (distal primers) regions to determine which polyadenylation site (PASI or PASII) is utilized in C2C12 myoblasts (MB) and C2C12 myotubes (MT). ( C ) qRT-PCR was used to quantify Pabpn1 levels using primers that recognize the Pabpn1 CDS or the distal Pabpn1 3′UTR. The levels of distal 3′UTR-containing transcripts is calculated relative to CDS-containing transcripts and normalized to Gapdh . As described in Materials and Methods, these data are presented as fold change relative to C2C12 myoblasts, for which the average value was set to 1.0. Data are mean ± SEM of n = 3 samples per cell type. ( D ) Representative northern blot using a radiolabeled probe recognizing both short (1.4 kb) and long (2.1kb) forms of the Pabpn1 transcript, corresponding to PASI and PASII utilization ( 22 ), respectively, assessing PAS usage in multiple mouse tissues and cell lines including C2C12 myoblasts (MB) and C2C12 myotubes (MT) that have been differentiated for 10 days. Because testis tissue contains high levels of predominantly short Pabpn1 transcript, this sample was underloaded to avoid strong signal. The 18S (1.9 kb) rRNA serves as a loading control. Data are representative of n = 4 independent biological replicates for C2C12 myotubes and n = 4 technical replicates for C2C12 myoblasts. ( E ) Quantification of Pabpn1 transcript levels from four independent northern blots of RNA prepared from C2C12 myoblasts (MB) and C2C12 myotubes (MT) was performed as described in Materials and Methods. Data were normalized to 18S rRNA as the loading control and are presented as fold change in Pabpn1 transcript levels relative to myoblast, which was set to 1.0. Data are mean ± SEM of n = 4 (* P
    Figure Legend Snippet: The long Pabpn1 3′UTR contains putative conserved cis- regulatory elements. ( A ) Schematic of the Pabpn1 transcript including a 5′ untranslated region (5′UTR), the coding DNA sequence ( Pabpn1 CDS), which encodes the PABPN1 open reading frame, and a 3′ untranslated region (3′UTR) which contains two polyadenylation signals (PASI, PASII) and multiple putative AU-rich elements (ARE1, ARE2, ARE3, ARE4). ( B ) Schematic of qRT-PCR strategy using primers that recognize the coding DNA sequence (CDS primers) and distal (distal primers) regions to determine which polyadenylation site (PASI or PASII) is utilized in C2C12 myoblasts (MB) and C2C12 myotubes (MT). ( C ) qRT-PCR was used to quantify Pabpn1 levels using primers that recognize the Pabpn1 CDS or the distal Pabpn1 3′UTR. The levels of distal 3′UTR-containing transcripts is calculated relative to CDS-containing transcripts and normalized to Gapdh . As described in Materials and Methods, these data are presented as fold change relative to C2C12 myoblasts, for which the average value was set to 1.0. Data are mean ± SEM of n = 3 samples per cell type. ( D ) Representative northern blot using a radiolabeled probe recognizing both short (1.4 kb) and long (2.1kb) forms of the Pabpn1 transcript, corresponding to PASI and PASII utilization ( 22 ), respectively, assessing PAS usage in multiple mouse tissues and cell lines including C2C12 myoblasts (MB) and C2C12 myotubes (MT) that have been differentiated for 10 days. Because testis tissue contains high levels of predominantly short Pabpn1 transcript, this sample was underloaded to avoid strong signal. The 18S (1.9 kb) rRNA serves as a loading control. Data are representative of n = 4 independent biological replicates for C2C12 myotubes and n = 4 technical replicates for C2C12 myoblasts. ( E ) Quantification of Pabpn1 transcript levels from four independent northern blots of RNA prepared from C2C12 myoblasts (MB) and C2C12 myotubes (MT) was performed as described in Materials and Methods. Data were normalized to 18S rRNA as the loading control and are presented as fold change in Pabpn1 transcript levels relative to myoblast, which was set to 1.0. Data are mean ± SEM of n = 4 (* P

    Techniques Used: Sequencing, Quantitative RT-PCR, Northern Blot

    Related Articles

    shRNA:

    Article Title: Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity
    Article Snippet: .. Binding reaction was setup with different concentrations of cell lysate, 1X Binding buffer (30 mM HEPES and 100 mM NaCl), [γ−32 P] ATP labelled shRNA probe (20,000 cpm per reaction) and 2 μg of salmon sperm DNA (Thermo Scientific). .. Unlabelled shRNA was added as cold probe to check specificity of the binding.

    Isolation:

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides
    Article Snippet: .. DNA Binding Assay DNA binding experiments were conducted with isolated EPS in combination with either chromosomal DNA from DK1622 or commercial salmon sperm DNA (2 Kb in size, 10 mg/ml, Invitrogen). .. 1 mg of isolated EPS was incubated with 0.1 mg chromosome DNA or salmon sperm DNA in 1 ml buffer at different pHs (0.15 M NaCl solution at pH 7.0; 0.1 M NaAc/HAc solution at pH 5.4; 0.01 M NaOH solution at pH 12.0), and denoted as ‘DNA+EPS’.

    Nick Translation:

    Article Title: Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis
    Article Snippet: .. BAC264k12 was labelled with digoxygenin (Boehringer) by nick translation and a probe consisting of labelled BAC DNA (200 ng), salmon sperm DNA (10 μg), Human Cot-1 DNA (5 μg; Gibco BRL) and chromosome 17 α-satellite DNA labelled with Spectrum Green (Vysis) was denatured and hybridized to denatured slides. ..

    Labeling:

    Article Title: Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response
    Article Snippet: .. Basic binding reactions were carried out on ice for 30 min and contained approximately 20 μg of nuclear extract (corrected so that each reaction contained the same amount of p53), 1 μg salmon sperm DNA, and 0.1 pmol 32 P-labeled (GIBCO/BRL 5′ DNA terminus labeling system) wild-type consensus sequence in a final volume of 20 μl. .. After incubation, the reaction mix was separated on a 4% polyacrylamide gel (200 volts for 1.5 hr), and the p53/consensus sequence complex was detected by autoradiography.

    Article Title: Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress
    Article Snippet: .. Radioactively labeled tRNA samples were mixed with 0.17 mg/ml salmon sperm DNA (Invitrogen), 0.17 mg/ml polyA (Sigma-Aldrich) in hybridization buffer (Sigma-Aldrich) and hybridized on the microarrays for 16 h at 60°C. ..

    DNA Binding Assay:

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides
    Article Snippet: .. DNA Binding Assay DNA binding experiments were conducted with isolated EPS in combination with either chromosomal DNA from DK1622 or commercial salmon sperm DNA (2 Kb in size, 10 mg/ml, Invitrogen). .. 1 mg of isolated EPS was incubated with 0.1 mg chromosome DNA or salmon sperm DNA in 1 ml buffer at different pHs (0.15 M NaCl solution at pH 7.0; 0.1 M NaAc/HAc solution at pH 5.4; 0.01 M NaOH solution at pH 12.0), and denoted as ‘DNA+EPS’.

    Mobility Shift:

    Article Title: Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein
    Article Snippet: .. For inhibition analysis, 2.7 nmol of either salmon sperm DNA (Invitrogen), nucleotides, or yeast tRNA (Sigma, St. Louis, MO) were added in addition to the standard mobility shift reaction mixtures. .. Surface Plasmon Resonance IsaB interactions with RNA, DNA, and dsDNA were analyzed using a BIAcore Model T100 (BIAcore International, Piscataway, NJ) following manufacturer's instructions.

    Inhibition:

    Article Title: Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein
    Article Snippet: .. For inhibition analysis, 2.7 nmol of either salmon sperm DNA (Invitrogen), nucleotides, or yeast tRNA (Sigma, St. Louis, MO) were added in addition to the standard mobility shift reaction mixtures. .. Surface Plasmon Resonance IsaB interactions with RNA, DNA, and dsDNA were analyzed using a BIAcore Model T100 (BIAcore International, Piscataway, NJ) following manufacturer's instructions.

    BAC Assay:

    Article Title: Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis
    Article Snippet: .. BAC264k12 was labelled with digoxygenin (Boehringer) by nick translation and a probe consisting of labelled BAC DNA (200 ng), salmon sperm DNA (10 μg), Human Cot-1 DNA (5 μg; Gibco BRL) and chromosome 17 α-satellite DNA labelled with Spectrum Green (Vysis) was denatured and hybridized to denatured slides. ..

    Sequencing:

    Article Title: Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response
    Article Snippet: .. Basic binding reactions were carried out on ice for 30 min and contained approximately 20 μg of nuclear extract (corrected so that each reaction contained the same amount of p53), 1 μg salmon sperm DNA, and 0.1 pmol 32 P-labeled (GIBCO/BRL 5′ DNA terminus labeling system) wild-type consensus sequence in a final volume of 20 μl. .. After incubation, the reaction mix was separated on a 4% polyacrylamide gel (200 volts for 1.5 hr), and the p53/consensus sequence complex was detected by autoradiography.

    Binding Assay:

    Article Title: Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response
    Article Snippet: .. Basic binding reactions were carried out on ice for 30 min and contained approximately 20 μg of nuclear extract (corrected so that each reaction contained the same amount of p53), 1 μg salmon sperm DNA, and 0.1 pmol 32 P-labeled (GIBCO/BRL 5′ DNA terminus labeling system) wild-type consensus sequence in a final volume of 20 μl. .. After incubation, the reaction mix was separated on a 4% polyacrylamide gel (200 volts for 1.5 hr), and the p53/consensus sequence complex was detected by autoradiography.

    Article Title: Gemfibrozil, a Lipid-lowering Drug, Induces Suppressor of Cytokine Signaling 3 in Glial Cells
    Article Snippet: .. The resultant nuclear extract pellet was frozen at −80 °C overnight, resuspended in a mixture of binding buffer, 10× Orange Loading Dye (Li-Cor Biosciences), a custom-designed fluorescent KLF4-specific probe (Li-Cor Biosciences), and salmon sperm DNA (Invitrogen) and electrophoresed on custom-cast 6% polyacrylamide gels. .. The shift was visualized under the Odyssey® Infrared Imaging System (Li-Cor).

    Article Title: Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity
    Article Snippet: .. Binding reaction was setup with different concentrations of cell lysate, 1X Binding buffer (30 mM HEPES and 100 mM NaCl), [γ−32 P] ATP labelled shRNA probe (20,000 cpm per reaction) and 2 μg of salmon sperm DNA (Thermo Scientific). .. Unlabelled shRNA was added as cold probe to check specificity of the binding.

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides
    Article Snippet: .. DNA Binding Assay DNA binding experiments were conducted with isolated EPS in combination with either chromosomal DNA from DK1622 or commercial salmon sperm DNA (2 Kb in size, 10 mg/ml, Invitrogen). .. 1 mg of isolated EPS was incubated with 0.1 mg chromosome DNA or salmon sperm DNA in 1 ml buffer at different pHs (0.15 M NaCl solution at pH 7.0; 0.1 M NaAc/HAc solution at pH 5.4; 0.01 M NaOH solution at pH 12.0), and denoted as ‘DNA+EPS’.

    Hybridization:

    Article Title: Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress
    Article Snippet: .. Radioactively labeled tRNA samples were mixed with 0.17 mg/ml salmon sperm DNA (Invitrogen), 0.17 mg/ml polyA (Sigma-Aldrich) in hybridization buffer (Sigma-Aldrich) and hybridized on the microarrays for 16 h at 60°C. ..

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    In vitro and in vivo assays to validate VSR activity. ( a ) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. ( b and c ) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. ( d ) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] <t>ATP.</t> 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. ( e )Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6 7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg 2 μg) of uncompetitive inhibitor. Salmon sperm <t>DNA</t> (2 μg) was used as non specific inhibitor in all binding reactions.
    Salmon Sperm Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of NFATc1/αA-bio and NFATc1/βC-bio proteins on cell death and the expression of Aicda and Prdm1 genes in murine WEHI 231 B lymphoma cells. (A) WEHI 231 B cells stably infected with retroviral vectors expressing BirA (WEHI-231), Bir A and NFATc1/αA-bio (NFATc1/αA), or BirA and NFATc1/βC-bio (NFATc1/βC) were stimulated with α-IgM or α-IgM α-IgM + αCD40 for 48 or 96 h. Apoptosis was determined by PI staining. MFI: Mean fluorescence intensity. (B) Wild-type (WT) WEHI cells (Co) or cells expressing NFATc1/αA-bio (blue) or NFATc1/βC-bio (red) were left unstimulated or stimulated for 6, 24, or 96 h with α-IgM. RNA was isolated and converted to cDNA libraries. DNA stretches of 50 bp were sequenced on a Illumina HiSeq2500 platform using the Truseq SBS kit-HS V3. Shown are the RNA reads (RPKM) from the Aicda and Prdm1 genes in the three types of WEHI cells. Results of one from two assays are shown. (C) Chromatin immuno precipitation (ChIP) assays for the binding of NFATc1-bio proteins to the Prdm1 gene in WEHI cells stimulated with T+I for 6 h. In the upper panel semi-quantitative PCR assays are shown for the detection of Prdm1 (and β -Actin ) DNA in chromatin precipitations. In the first three lanes, chromatin from WEHI cells transfected with BirA alone, with NFATc1/αA-bio (+BirA) or NFATc1/βC-bio (+BirA) was precipitated with <t>streptavidin-agarose</t> beads. In the next lanes, chromatin was precipitated with Abs specific for histone H3, NFATc1 (7A6), and immunoglobulin. In the last two lanes, DNA input and H 2 O controls are shown. One typical assay from three assays is shown. In the lower panel the enrichment of β -Actin, Rcan1, Prdm1, Il2 , and Ppp3ca DNAs precipitated with streptavidin beads from WEHI cells expressing either NFATc1/αA-bio or NFATc1/βC-bio is shown. Mean values of three assays are shown. (D) ChIP assays indicating histone modifications at the Prdm1 promoter. ChIP assays were performed with chromatin from WEHI cells overexpressing NFATc1/αA-bio (+BirA) (open bars) or NFATc1/βC-bio (+BirA) proteins (gray bars) using Abs directed the histone modifications H3K9me3 and H3K9ac, respectively. In semi-quantitative PCR assays, primers detecting the Prdm1 ) were used. Mean values of three assays are shown.
    Streptavidin Agarose Resin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In vitro and in vivo assays to validate VSR activity. ( a ) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. ( b and c ) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. ( d ) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] ATP. 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. ( e )Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6 7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg 2 μg) of uncompetitive inhibitor. Salmon sperm DNA (2 μg) was used as non specific inhibitor in all binding reactions.

    Journal: Scientific Reports

    Article Title: Analysis of chikungunya virus proteins reveals that non-structural proteins nsP2 and nsP3 exhibit RNA interference (RNAi) suppressor activity

    doi: 10.1038/srep38065

    Figure Lengend Snippet: In vitro and in vivo assays to validate VSR activity. ( a ) Western blotting to show changes in GFP levels upon transfection with nsP2 and nsP3. Sf21 sensor cell line was transfected with VSRs and western blotting was done using anti-GFP antibody. GADPH was used as housekeeping control. ( b and c ) CHIKV nsP2 and nsP3 show RNAi suppressor activity in in vivo system. Transgenic Nicotiana leaves with GFPshRNA stably integrated were infiltrated with VSR expressing Agrobacterium cultures and checked for GFP reversion under UV transilluminator. FHVB2 was used as positive control and mutated FHVB2 was the negative control. FHVB2M shows necrosis marks due to infiltration, but no GFP reversion was seen. ( d ) Northern blotting to show changes in GFP mRNA and small RNA levels upon VSR infiltration in Nicotiana leaves. RNA isolated from infiltrated leaves was used to detect GFP mRNA levels using GFPshRNA oligonucleotide end labelled with [γ32P] ATP. 18 S was used as housekeeping control. GFP small RNA population was detected by northern blotting using 700 bp DIG labelled GFP probe. 28SrRNA was used as house keeping control. ( e )Electrophoretic mobility shift assay (EMSA) using labeled GFPshRNA probe and VSR transfected Sf21 cell lysate. GFPshRNA oligonucleotide was end-labelled with [γ32P] ATP and mixed with different concentrations of VSR transfected Sf21 cell lysate. Lane 1: Free shRNA probe; lane 2, 3, 4, 5: Different concentrations of nsP3 (30 μg, 20 μg) and nsP2 (30 μg, 20 μg) transfected Sf21 lysate respectively; lane 6 7: nsP3 and nsP2 transfected Sf21 cell lysate with 100 fold unlabelled GFPshRNA probe; lane 8, 9 10: binding of untransfected Sf21 cells to GFPshRNA in the absence and presence (1 μg 2 μg) of uncompetitive inhibitor. Salmon sperm DNA (2 μg) was used as non specific inhibitor in all binding reactions.

    Article Snippet: Binding reaction was setup with different concentrations of cell lysate, 1X Binding buffer (30 mM HEPES and 100 mM NaCl), [γ−32 P] ATP labelled shRNA probe (20,000 cpm per reaction) and 2 μg of salmon sperm DNA (Thermo Scientific).

    Techniques: In Vitro, In Vivo, Activity Assay, Western Blot, Transfection, Transgenic Assay, Stable Transfection, Expressing, Positive Control, Negative Control, Northern Blot, Isolation, Electrophoretic Mobility Shift Assay, Labeling, shRNA, Binding Assay

    TLs manifest transcription progression, including co-transcriptional splicing, and dynamically modify harboring chromosomal loci A , Successive labeling of Tg TLs with probes for 5’ and 3’ exons. A1, Distribution of 11 5’ exons ( green ) and 15 3’ exons ( red ) of the Tg gene chosen for RNA-FISH. A2,3, Schematics showing changes in the composition of nRNAs during transcription progression: nRNAs of the first half of a TL contain only “ green ” 5’ exons; in the second half they also include “ red ” 3’ exons (A2). Correspondingly, “ green ” exons label transcripts along the whole TL and “ red ” exons label only transcripts of the TL second half (A3). A4, Examples of Tg TLs labeling after RNA-FISH with probes described in A1. Probe for the 5’ exons labels the entire TL, whereas the probe for 3’ exons labels second half of the TL. Arrows point at the TL regions labeled with only 5’ probe. B , Sequential labeling of Tg TLs with genomic probes highlighting introns. B1, coverage of the Tg gene with two overlapping BACs, for 5’ ( green ) and 3’ ( red ) gene halves. B2,3, Schematics explaining changes in nRNA labeling with probes highlighting introns during transcription progression: “ green ” 5’ introns are gradually spliced out and replaced by “ red ” 3’ introns (B2). Correspondingly, “ green ” introns label transcripts of the first and “ red ” exons of the second TL halves with some overlap ( yellow , B3). B4, Example of Tg TLs after RNA-FISH with two BAC probes described in B1. BAC probes highlighting 5’ ( green ) and 3’ ( red ) introns sequentially label the TLs as a result of co-transcriptional splicing. In addition, since the 5’ probe includes 5’ exons, it also faintly labeled the second half of the loop ( arrows ). The region hybridized by both overlapping BACs is marked with arrowheads. ex , exons; int , introns. Black arrows indicate direction of transcription. Scale bars, 2 µm C , TLs formed by other long highly expressed genes exhibit co-transcriptional splicing. RNA-FISH with BAC probes encompassing 5’ ( green ) and 3’ ( red ) regions of the genes (see SI Table S3 for the probes). Projections of confocal sections through 1 - 2 µm . Scale bars, 2 µm D , Expressed genes expand from the harboring loci and separate their flanks. Distances between 5’ ( green ) and 3’ ( red ) flanking regions of the Tg (D1) and Ttn (D2) genes are larger in cells expressing ( left panels ) compared to control cells not expressing the genes ( right panels ), as shown by schematics at the bottom. Boxplots depicting the 3D distance between the flanking regions in expressing and not-expressing cells are shown on the right. The median inter-flank distance for Tg in thyrocytes is 2.3-fold larger than in control epithelial cells (703 nm versus 311 nm). The median inter-flank distance for Ttn in myotubes is 1.7-fold larger than in control myoblasts (1,104 nm versus 634 nm). FISH with simultaneous detection of DNA and RNA signals. Projections of confocal sections through 1 - 3 µm . Scale bars, 2 µm

    Journal: bioRxiv

    Article Title: SPATIAL ORGANIZATION OF TRANSCRIBED EUKARYOTIC GENES

    doi: 10.1101/2020.05.20.106591

    Figure Lengend Snippet: TLs manifest transcription progression, including co-transcriptional splicing, and dynamically modify harboring chromosomal loci A , Successive labeling of Tg TLs with probes for 5’ and 3’ exons. A1, Distribution of 11 5’ exons ( green ) and 15 3’ exons ( red ) of the Tg gene chosen for RNA-FISH. A2,3, Schematics showing changes in the composition of nRNAs during transcription progression: nRNAs of the first half of a TL contain only “ green ” 5’ exons; in the second half they also include “ red ” 3’ exons (A2). Correspondingly, “ green ” exons label transcripts along the whole TL and “ red ” exons label only transcripts of the TL second half (A3). A4, Examples of Tg TLs labeling after RNA-FISH with probes described in A1. Probe for the 5’ exons labels the entire TL, whereas the probe for 3’ exons labels second half of the TL. Arrows point at the TL regions labeled with only 5’ probe. B , Sequential labeling of Tg TLs with genomic probes highlighting introns. B1, coverage of the Tg gene with two overlapping BACs, for 5’ ( green ) and 3’ ( red ) gene halves. B2,3, Schematics explaining changes in nRNA labeling with probes highlighting introns during transcription progression: “ green ” 5’ introns are gradually spliced out and replaced by “ red ” 3’ introns (B2). Correspondingly, “ green ” introns label transcripts of the first and “ red ” exons of the second TL halves with some overlap ( yellow , B3). B4, Example of Tg TLs after RNA-FISH with two BAC probes described in B1. BAC probes highlighting 5’ ( green ) and 3’ ( red ) introns sequentially label the TLs as a result of co-transcriptional splicing. In addition, since the 5’ probe includes 5’ exons, it also faintly labeled the second half of the loop ( arrows ). The region hybridized by both overlapping BACs is marked with arrowheads. ex , exons; int , introns. Black arrows indicate direction of transcription. Scale bars, 2 µm C , TLs formed by other long highly expressed genes exhibit co-transcriptional splicing. RNA-FISH with BAC probes encompassing 5’ ( green ) and 3’ ( red ) regions of the genes (see SI Table S3 for the probes). Projections of confocal sections through 1 - 2 µm . Scale bars, 2 µm D , Expressed genes expand from the harboring loci and separate their flanks. Distances between 5’ ( green ) and 3’ ( red ) flanking regions of the Tg (D1) and Ttn (D2) genes are larger in cells expressing ( left panels ) compared to control cells not expressing the genes ( right panels ), as shown by schematics at the bottom. Boxplots depicting the 3D distance between the flanking regions in expressing and not-expressing cells are shown on the right. The median inter-flank distance for Tg in thyrocytes is 2.3-fold larger than in control epithelial cells (703 nm versus 311 nm). The median inter-flank distance for Ttn in myotubes is 1.7-fold larger than in control myoblasts (1,104 nm versus 634 nm). FISH with simultaneous detection of DNA and RNA signals. Projections of confocal sections through 1 - 3 µm . Scale bars, 2 µm

    Article Snippet: A typical hybridization mixture for 2D FISH with a BAC derived probe and a chromosome paint was set-up as follows: 15 µl chromosome paint–BIO, 25 µl BAC–DIG, 20 µl Cot-1 DNA (Thermo Fisher), 2 µl salmon sperm DNA (Thermo Fisher), 300 µl pre-cooled (−20 °C) EtOH.

    Techniques: Labeling, Fluorescence In Situ Hybridization, BAC Assay, Expressing

    Site transfer probabilities and kinetic activity of hOGG1 in the presence of variable cosolutes. ( A ) The multistep search and repair pathway of DNA glycosylases involves associative and dissociative translocation along the DNA chain (see text). ( B ) Phosphorimages of the products derived from reaction of hOGG1 with two 90mer substrates (S20 oxoG and S10 oxoG ) with two oxo G residues positioned 10 and 20bp apart. Solution conditions and co-solutes are indicated under each lane of the gel images. ( C ) The transfer probabilities between oxo G sites in S10 oxoG and S20 oxoG in the presence of variable cosolutes using 30 mM (red) and 150 mM salt (black) ( T = 37°C). ( D ) Effects of salt, 20% PEG 8K, and salDNA on the observed rate of 8-oxoG excision from S20 oxoG . Error is derived from 3 independent trials of each condition.

    Journal: Biochemistry

    Article Title: Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG

    doi: 10.1021/acs.biochem.6b00482

    Figure Lengend Snippet: Site transfer probabilities and kinetic activity of hOGG1 in the presence of variable cosolutes. ( A ) The multistep search and repair pathway of DNA glycosylases involves associative and dissociative translocation along the DNA chain (see text). ( B ) Phosphorimages of the products derived from reaction of hOGG1 with two 90mer substrates (S20 oxoG and S10 oxoG ) with two oxo G residues positioned 10 and 20bp apart. Solution conditions and co-solutes are indicated under each lane of the gel images. ( C ) The transfer probabilities between oxo G sites in S10 oxoG and S20 oxoG in the presence of variable cosolutes using 30 mM (red) and 150 mM salt (black) ( T = 37°C). ( D ) Effects of salt, 20% PEG 8K, and salDNA on the observed rate of 8-oxoG excision from S20 oxoG . Error is derived from 3 independent trials of each condition.

    Article Snippet: Salmon sperm DNA (salDNA) was purchased from ThermoFisher Scientific.

    Techniques: Activity Assay, Translocation Assay, Derivative Assay

    Summary of the effects of salt, molecular crowding, and bulk DNA on each measured thermodynamic and kinetic parameter. ( A ) The salt effect (X 150 /X LS ) is indicated for each solution condition we have explored. The salt effect is defined as the value of a given measurement ( K D , P trans or V 0 /[E]) at 150 mM salt (X 150 ) divided by the same measurement at low salt (X LS ). This panel compares the salt effect in dilute buffered solution as compared to when 20% PEG8K, 1 mM bulk DNA, or both are present. Salt effects falling below the dashed line are reduced by high ionic strength and those above are enhanced. hUNG and hOGG1 respond differently to the introduction of salt with respect to non-specific DNA binding ( K D N ) and kinetic activity in the presence of crowder and salDNA. ( B ) General model for the effects of molecular crowders (orange lines) and bulk DNA (purple bars) on DNA translocation at a physiological salt concentration. The image is drawn to scale using a DNA duplex of with a 2 nm diameter as a scale reference. Dashed lines depict the depletion layer ( R g PEG .

    Journal: Biochemistry

    Article Title: Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG

    doi: 10.1021/acs.biochem.6b00482

    Figure Lengend Snippet: Summary of the effects of salt, molecular crowding, and bulk DNA on each measured thermodynamic and kinetic parameter. ( A ) The salt effect (X 150 /X LS ) is indicated for each solution condition we have explored. The salt effect is defined as the value of a given measurement ( K D , P trans or V 0 /[E]) at 150 mM salt (X 150 ) divided by the same measurement at low salt (X LS ). This panel compares the salt effect in dilute buffered solution as compared to when 20% PEG8K, 1 mM bulk DNA, or both are present. Salt effects falling below the dashed line are reduced by high ionic strength and those above are enhanced. hUNG and hOGG1 respond differently to the introduction of salt with respect to non-specific DNA binding ( K D N ) and kinetic activity in the presence of crowder and salDNA. ( B ) General model for the effects of molecular crowders (orange lines) and bulk DNA (purple bars) on DNA translocation at a physiological salt concentration. The image is drawn to scale using a DNA duplex of with a 2 nm diameter as a scale reference. Dashed lines depict the depletion layer ( R g PEG .

    Article Snippet: Salmon sperm DNA (salDNA) was purchased from ThermoFisher Scientific.

    Techniques: Binding Assay, Activity Assay, Translocation Assay, Concentration Assay

    Effect of NFATc1/αA-bio and NFATc1/βC-bio proteins on cell death and the expression of Aicda and Prdm1 genes in murine WEHI 231 B lymphoma cells. (A) WEHI 231 B cells stably infected with retroviral vectors expressing BirA (WEHI-231), Bir A and NFATc1/αA-bio (NFATc1/αA), or BirA and NFATc1/βC-bio (NFATc1/βC) were stimulated with α-IgM or α-IgM α-IgM + αCD40 for 48 or 96 h. Apoptosis was determined by PI staining. MFI: Mean fluorescence intensity. (B) Wild-type (WT) WEHI cells (Co) or cells expressing NFATc1/αA-bio (blue) or NFATc1/βC-bio (red) were left unstimulated or stimulated for 6, 24, or 96 h with α-IgM. RNA was isolated and converted to cDNA libraries. DNA stretches of 50 bp were sequenced on a Illumina HiSeq2500 platform using the Truseq SBS kit-HS V3. Shown are the RNA reads (RPKM) from the Aicda and Prdm1 genes in the three types of WEHI cells. Results of one from two assays are shown. (C) Chromatin immuno precipitation (ChIP) assays for the binding of NFATc1-bio proteins to the Prdm1 gene in WEHI cells stimulated with T+I for 6 h. In the upper panel semi-quantitative PCR assays are shown for the detection of Prdm1 (and β -Actin ) DNA in chromatin precipitations. In the first three lanes, chromatin from WEHI cells transfected with BirA alone, with NFATc1/αA-bio (+BirA) or NFATc1/βC-bio (+BirA) was precipitated with streptavidin-agarose beads. In the next lanes, chromatin was precipitated with Abs specific for histone H3, NFATc1 (7A6), and immunoglobulin. In the last two lanes, DNA input and H 2 O controls are shown. One typical assay from three assays is shown. In the lower panel the enrichment of β -Actin, Rcan1, Prdm1, Il2 , and Ppp3ca DNAs precipitated with streptavidin beads from WEHI cells expressing either NFATc1/αA-bio or NFATc1/βC-bio is shown. Mean values of three assays are shown. (D) ChIP assays indicating histone modifications at the Prdm1 promoter. ChIP assays were performed with chromatin from WEHI cells overexpressing NFATc1/αA-bio (+BirA) (open bars) or NFATc1/βC-bio (+BirA) proteins (gray bars) using Abs directed the histone modifications H3K9me3 and H3K9ac, respectively. In semi-quantitative PCR assays, primers detecting the Prdm1 ) were used. Mean values of three assays are shown.

    Journal: Frontiers in Immunology

    Article Title: Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation

    doi: 10.3389/fimmu.2018.00032

    Figure Lengend Snippet: Effect of NFATc1/αA-bio and NFATc1/βC-bio proteins on cell death and the expression of Aicda and Prdm1 genes in murine WEHI 231 B lymphoma cells. (A) WEHI 231 B cells stably infected with retroviral vectors expressing BirA (WEHI-231), Bir A and NFATc1/αA-bio (NFATc1/αA), or BirA and NFATc1/βC-bio (NFATc1/βC) were stimulated with α-IgM or α-IgM α-IgM + αCD40 for 48 or 96 h. Apoptosis was determined by PI staining. MFI: Mean fluorescence intensity. (B) Wild-type (WT) WEHI cells (Co) or cells expressing NFATc1/αA-bio (blue) or NFATc1/βC-bio (red) were left unstimulated or stimulated for 6, 24, or 96 h with α-IgM. RNA was isolated and converted to cDNA libraries. DNA stretches of 50 bp were sequenced on a Illumina HiSeq2500 platform using the Truseq SBS kit-HS V3. Shown are the RNA reads (RPKM) from the Aicda and Prdm1 genes in the three types of WEHI cells. Results of one from two assays are shown. (C) Chromatin immuno precipitation (ChIP) assays for the binding of NFATc1-bio proteins to the Prdm1 gene in WEHI cells stimulated with T+I for 6 h. In the upper panel semi-quantitative PCR assays are shown for the detection of Prdm1 (and β -Actin ) DNA in chromatin precipitations. In the first three lanes, chromatin from WEHI cells transfected with BirA alone, with NFATc1/αA-bio (+BirA) or NFATc1/βC-bio (+BirA) was precipitated with streptavidin-agarose beads. In the next lanes, chromatin was precipitated with Abs specific for histone H3, NFATc1 (7A6), and immunoglobulin. In the last two lanes, DNA input and H 2 O controls are shown. One typical assay from three assays is shown. In the lower panel the enrichment of β -Actin, Rcan1, Prdm1, Il2 , and Ppp3ca DNAs precipitated with streptavidin beads from WEHI cells expressing either NFATc1/αA-bio or NFATc1/βC-bio is shown. Mean values of three assays are shown. (D) ChIP assays indicating histone modifications at the Prdm1 promoter. ChIP assays were performed with chromatin from WEHI cells overexpressing NFATc1/αA-bio (+BirA) (open bars) or NFATc1/βC-bio (+BirA) proteins (gray bars) using Abs directed the histone modifications H3K9me3 and H3K9ac, respectively. In semi-quantitative PCR assays, primers detecting the Prdm1 ) were used. Mean values of three assays are shown.

    Article Snippet: Chromatin in 300 µl sonication buffer was incubated with 25 µl of streptavidin agarose resin (ThermoScientific, #20347; 50% slurry saturated with Salmon Sperm DNA and FGEL), or 2 µl NFATc1 Ab (7A6, BD 556609), or 2 µl Ig Ab, or 2 µl H3 Ab (CellSignaling; #4620s), or 2 µl H3K9me3 Ab (abcam ab8898), or 2 µl H3K9ac Ab (Merck 07-352) at 4°C o.n.

    Techniques: Expressing, Stable Transfection, Infection, Staining, Fluorescence, Isolation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Transfection