pfastbacht a  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pfastbacht a
    PCR products verifying the recombinant plasmids pFastBacHT A-Der f 1, pFastBacHT A-Der f 2, and pFast-BacHT A-Der f 4. Lanes 1–8, Der f 1; lanes 9–16, Der f 2; lanes 17–24, Der f 4; lane M1, DNA marker DL 2000; lane M 2 , λ-Hind III DNA marker
    Pfastbacht A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 19425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 19425 article reviews
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    pfastbacht a - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Expression of recombinant allergen, Der f 1, Der f 2 and Der f 4 using baculovirus-insect cell systems"

    Article Title: Expression of recombinant allergen, Der f 1, Der f 2 and Der f 4 using baculovirus-insect cell systems

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2018.79005

    PCR products verifying the recombinant plasmids pFastBacHT A-Der f 1, pFastBacHT A-Der f 2, and pFast-BacHT A-Der f 4. Lanes 1–8, Der f 1; lanes 9–16, Der f 2; lanes 17–24, Der f 4; lane M1, DNA marker DL 2000; lane M 2 , λ-Hind III DNA marker
    Figure Legend Snippet: PCR products verifying the recombinant plasmids pFastBacHT A-Der f 1, pFastBacHT A-Der f 2, and pFast-BacHT A-Der f 4. Lanes 1–8, Der f 1; lanes 9–16, Der f 2; lanes 17–24, Der f 4; lane M1, DNA marker DL 2000; lane M 2 , λ-Hind III DNA marker

    Techniques Used: Polymerase Chain Reaction, Recombinant, Marker

    2) Product Images from "GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics"

    Article Title: GPCR-induced calcium transients trigger nuclear actin assembly for chromatin dynamics

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13322-y

    Calcium transients induce nuclear F-actin assembly. a Representative images of NIH3T3 cells stably expressing nAC-GFP after A23187 stimulation. In total, 73.6% ± 7.6% (s.e.m.) cells exhibited NAA. Each experiment included three samples in parallel, and about 30 cells were recorded and counted each time. Experiments were repeated three times ( n = 3). Scale bar: 10 μm. b NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum or A23187 (750 nM) after pre-treatment with or without LPA receptor inhibitor Ki16425 (20 μM) at the confocal microscope. n = 4 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. c NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum after pre-treatment with or without BAPTA-AM (10 μM). n = 3 independent experiments. Two-sided Student's t test, ** p ≤ 0.01. d NIH3T3 cells stably expressing nAC-GFP were stimulated with LPA (20 μM), thrombin (0.2 U/mL), or ATP (10 μM). n = 3 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. Error bars: +s.e.m. e Representative images of NIH3T3 cells stably expressing nAC-GFP after thrombin stimulation. Experiments were performed three times, and about 30 cells were recorded each time. Eighty percent of the cells were positive for NAA as shown in panel d . Scale bar: 10 μm. Source data are provided as a Source Data file
    Figure Legend Snippet: Calcium transients induce nuclear F-actin assembly. a Representative images of NIH3T3 cells stably expressing nAC-GFP after A23187 stimulation. In total, 73.6% ± 7.6% (s.e.m.) cells exhibited NAA. Each experiment included three samples in parallel, and about 30 cells were recorded and counted each time. Experiments were repeated three times ( n = 3). Scale bar: 10 μm. b NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum or A23187 (750 nM) after pre-treatment with or without LPA receptor inhibitor Ki16425 (20 μM) at the confocal microscope. n = 4 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. c NIH3T3 cells stably expressing nAC-GFP were stimulated with 20% serum after pre-treatment with or without BAPTA-AM (10 μM). n = 3 independent experiments. Two-sided Student's t test, ** p ≤ 0.01. d NIH3T3 cells stably expressing nAC-GFP were stimulated with LPA (20 μM), thrombin (0.2 U/mL), or ATP (10 μM). n = 3 independent experiments. One-way ANOVA test, **** p ≤ 0.0001. Error bars: +s.e.m. e Representative images of NIH3T3 cells stably expressing nAC-GFP after thrombin stimulation. Experiments were performed three times, and about 30 cells were recorded each time. Eighty percent of the cells were positive for NAA as shown in panel d . Scale bar: 10 μm. Source data are provided as a Source Data file

    Techniques Used: Stable Transfection, Expressing, Microscopy

    3) Product Images from "Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity"

    Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068648

    DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).
    Figure Legend Snippet: DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction

    4) Product Images from "Antioxidant Activity and Acetylcholinesterase Inhibition of Grape Skin Anthocyanin (GSA)"

    Article Title: Antioxidant Activity and Acetylcholinesterase Inhibition of Grape Skin Anthocyanin (GSA)

    Journal: Molecules

    doi: 10.3390/molecules19079403

    Protective effect of GSA on hydroxyl radical-mediated pBR322 DNA strand breaks. ( A ) GSA ( B ) Vitamin C. Lane 1: normal DNA control; lane 2: FeSO 4 + H 2 O 2 (DNA damage control); lane: 3–5: FeSO 4 + H 2 O 2 + DNA in the presence of GSA (125, 250 and 500 µg/mL, respectively).
    Figure Legend Snippet: Protective effect of GSA on hydroxyl radical-mediated pBR322 DNA strand breaks. ( A ) GSA ( B ) Vitamin C. Lane 1: normal DNA control; lane 2: FeSO 4 + H 2 O 2 (DNA damage control); lane: 3–5: FeSO 4 + H 2 O 2 + DNA in the presence of GSA (125, 250 and 500 µg/mL, respectively).

    Techniques Used:

    5) Product Images from "Global and Quantitative Profiling of Polyadenylated RNAs Using PAS-seq"

    Article Title: Global and Quantitative Profiling of Polyadenylated RNAs Using PAS-seq

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-971-0_16

    PAS-seq library. 10 % of the second-round PCR reaction (Subheading 3.6) is resolved on a 2 % agarose gel to check the size of DNA fragments
    Figure Legend Snippet: PAS-seq library. 10 % of the second-round PCR reaction (Subheading 3.6) is resolved on a 2 % agarose gel to check the size of DNA fragments

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    6) Product Images from "Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes"

    Article Title: Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes

    Journal: The Journal of Organic Chemistry

    doi: 10.1021/acs.joc.6b00369

    Recognition of DNA hairpins using Invader probes. (a) Illustration of recognition process; (b) sequences and thermal denaturation temperatures of DNA hairpins with isosequential ( DH1 ) or nonisosequential stems ( DH2 – DH7 ); underlined nucleotides indicate sequence deviations relative to probes; (c) representative electrophoretograms of recognition of DH1 using 1- to 500-fold molar excess of Y1 : Y3 or DY1 : DY4 ; (d) dose–response curves (average of at least three independent experiments; error bars represent standard deviation); (e) electrophoretograms illustrating incubation of DH1 – DH7 with 200-fold molar excess of X1 : X3 , Y1 : Y3 , or DY1 : DY4 . Experimental conditions for electrophoretic mobility shift assay: separately preannealed targets (34.4 nM) and probes (variable concentrations) were incubated for 12–16 h at ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 10% sucrose, 1.4 mM spermine tetrahydrochloride, pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h, ∼4 °C) using 0.5× TBE as a running buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.
    Figure Legend Snippet: Recognition of DNA hairpins using Invader probes. (a) Illustration of recognition process; (b) sequences and thermal denaturation temperatures of DNA hairpins with isosequential ( DH1 ) or nonisosequential stems ( DH2 – DH7 ); underlined nucleotides indicate sequence deviations relative to probes; (c) representative electrophoretograms of recognition of DH1 using 1- to 500-fold molar excess of Y1 : Y3 or DY1 : DY4 ; (d) dose–response curves (average of at least three independent experiments; error bars represent standard deviation); (e) electrophoretograms illustrating incubation of DH1 – DH7 with 200-fold molar excess of X1 : X3 , Y1 : Y3 , or DY1 : DY4 . Experimental conditions for electrophoretic mobility shift assay: separately preannealed targets (34.4 nM) and probes (variable concentrations) were incubated for 12–16 h at ambient temperature in 1X HEPES buffer (50 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 10% sucrose, 1.4 mM spermine tetrahydrochloride, pH 7.2) and then resolved on 16% nondenaturing PAGE (70 V, 2.5 h, ∼4 °C) using 0.5× TBE as a running buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA); DIG: digoxigenin.

    Techniques Used: Sequencing, Standard Deviation, Incubation, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Sustained Release of Hydrogen Sulfide (H2S) from Poly(lactic acid) Functionalized 4-hydroxythiobenzamide Microparticles to Protect against Oxidative Damage"

    Article Title: Sustained Release of Hydrogen Sulfide (H2S) from Poly(lactic acid) Functionalized 4-hydroxythiobenzamide Microparticles to Protect against Oxidative Damage

    Journal: Annals of biomedical engineering

    doi: 10.1007/s10439-019-02270-9

    Assessment of ROS protection by PLA-4HTB microparticles. Median dihydroethidium (DHE) fluorescence was measured in HEK 293 cells after incubation with different treatments with and without particles, followed by incubation with 600 μM CoCl 2 for 24h. Cells were analyzed using flow cytometry and statistically analyzed using a one-way ANOVA followed by a Tukey’s comparison test. n = 4, **p
    Figure Legend Snippet: Assessment of ROS protection by PLA-4HTB microparticles. Median dihydroethidium (DHE) fluorescence was measured in HEK 293 cells after incubation with different treatments with and without particles, followed by incubation with 600 μM CoCl 2 for 24h. Cells were analyzed using flow cytometry and statistically analyzed using a one-way ANOVA followed by a Tukey’s comparison test. n = 4, **p

    Techniques Used: Proximity Ligation Assay, Fluorescence, Incubation, Flow Cytometry

    8) Product Images from "Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA"

    Article Title: Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5? Capped 2?-O Unmethylated RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.00883-13

    Ifit1 preferentially binds to virus RNA lacking 2′-O methylation. (A) Electrophoretic mobility shift of biotin-labeled RNA (JEV 5′-terminal 200 nucleotides) with recombinant Ifit1. The presence or absence of a 5′ cap and 2′-O
    Figure Legend Snippet: Ifit1 preferentially binds to virus RNA lacking 2′-O methylation. (A) Electrophoretic mobility shift of biotin-labeled RNA (JEV 5′-terminal 200 nucleotides) with recombinant Ifit1. The presence or absence of a 5′ cap and 2′-O

    Techniques Used: Methylation, Electrophoretic Mobility Shift Assay, Labeling, Recombinant

    9) Product Images from "A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy"

    Article Title: A Long Non-Coding RNA Defines an Epigenetic Checkpoint in Cardiac Hypertrophy

    Journal: Nature medicine

    doi: 10.1038/nm.4179

    Characterization of Chaer motif for PRC2 interaction ( a ) Predicted secondary structures of a 66-mer motif in mouse mChaer , a 67-mer motif in rat rChaer , a 62-mer motif in human hCHAER and an 89-mer motif in mouse mHotair . The unpaired loops with similar pattern were highlighted by gray background, and the paired single nucleotide variations in stems between mouse and rat Chaer were highlighted with red boxes. ( b ) Validation of the direct binding between the 66-mer mChaer motif and recombinant Ezh2 by RNA electrophoretic mobility shift assay (EMSA). ( c ) RNA EMSA using labeled Chaer and unlabeled Chaer or Hotair (left). Dissociation constants were calculated by the concentration of unlabeled Chaer or Hotair causing 50% dissociation of the Ezh2- Chaer complex (right). ( d ) Interaction propensity for Ezh2 binding with Chaer 66-mer motif, Hotair 89-mer motif, Chaer 201-300-nt fragment and 5.8 S rRNA predicted by CatRAPID. Arrowheads highlights the predicted RNA-binding sites of Ezh2.
    Figure Legend Snippet: Characterization of Chaer motif for PRC2 interaction ( a ) Predicted secondary structures of a 66-mer motif in mouse mChaer , a 67-mer motif in rat rChaer , a 62-mer motif in human hCHAER and an 89-mer motif in mouse mHotair . The unpaired loops with similar pattern were highlighted by gray background, and the paired single nucleotide variations in stems between mouse and rat Chaer were highlighted with red boxes. ( b ) Validation of the direct binding between the 66-mer mChaer motif and recombinant Ezh2 by RNA electrophoretic mobility shift assay (EMSA). ( c ) RNA EMSA using labeled Chaer and unlabeled Chaer or Hotair (left). Dissociation constants were calculated by the concentration of unlabeled Chaer or Hotair causing 50% dissociation of the Ezh2- Chaer complex (right). ( d ) Interaction propensity for Ezh2 binding with Chaer 66-mer motif, Hotair 89-mer motif, Chaer 201-300-nt fragment and 5.8 S rRNA predicted by CatRAPID. Arrowheads highlights the predicted RNA-binding sites of Ezh2.

    Techniques Used: Binding Assay, Recombinant, Electrophoretic Mobility Shift Assay, Labeling, Concentration Assay, RNA Binding Assay

    10) Product Images from "The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis"

    Article Title: The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072793

    Multiple protein complexes are associated with Ast 3′-UTR 242–483 . RNA EMSA analysis of Ast 3′-UTR 242–483 RNA incubated with shrimp hemocyte extracts shows that four protein complexes are associated with Ast 3′-UTR 242–483 (C1–C4).
    Figure Legend Snippet: Multiple protein complexes are associated with Ast 3′-UTR 242–483 . RNA EMSA analysis of Ast 3′-UTR 242–483 RNA incubated with shrimp hemocyte extracts shows that four protein complexes are associated with Ast 3′-UTR 242–483 (C1–C4).

    Techniques Used: AST Assay, Incubation

    11) Product Images from "YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1"

    Article Title: YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201411047

    YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1 - and XIAP -specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1 /YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.
    Figure Legend Snippet: YB-1 regulates G3BP1 translation through the G3BP1 5′ UTR. (A) mRNA transcripts bound to YB-1 were riboimmunoprecipitated (RIPed) using anti–YB-1 antibodies or normal rabbit serum (NRS) from siControl and siYB-1 kd cell lysates. Captured mRNAs were reverse-transcribed and PCR amplified using primers specific for G3BP1 or XIAP as a control. (B) YB-1–bound mRNAs were RIPed using anti–YB-1 or control anti-GRB2 antibodies from polysomes prepared from vehicle alone and arsenite-treated U2OS cells, and subjected to semiquantitative RT-PCR using G3BP1 - and XIAP -specific primers. (C) Constructs containing 5′ UTR sequences of G3BP1 (black) or β-Globin (gray) fused in frame to Luciferase were used for in vitro coupled transcription translation. Increasing concentrations of recombinant YB-1 were added to the assay mixture, and luciferase activity was measured. Error bars indicate SD. (D) RNA EMSA analysis to measure direct binding of YB-1 to the full-length G3BP1 5′ UTR. Biotin-labeled full-length G3BP1 5′ UTR mixed with recombinant GST-YB-1 was subjected to EMSA. The arrowhead indicates a probe mobility shift in the presence of 0.4 µg of GST-YB-1, and enhanced intensity at 0.8 µg of GST-YB-1. A 200-fold molar excess concentration of unlabeled full-length G3BP1 5′ UTR was added to demonstrate specificity of 5′ UTR G3BP1 /YB-1 complex formation. As a control, recombinant GST was used in place of GST-YB-1. The broken line indicates that intervening lanes have been spliced out. (E) The full-length 5′ UTR G3BP1 (FL, 1–171) or deletion mutants (M1, Δ105–112; M2, Δ141–171; M3, Δ99–171; and M4, Δ141–171) were cloned in frame with Luciferase and used for in vitro coupled transcription/translation assays ±0.5 pmol YB-1 as described in C. Error bars indicate SD. (F) RNA EMSA showing that YB-1 binds to the full-length (FL, 1–171) G3BP1 5′ UTR but not M3 and M4 mutants. (G) Biotin end-tagged full length or the indicated deletion mutants of the G3BP1 5′ UTR were subjected to RNA affinity chromatography from U2OS lysates using Streptavidin beads. Affinity-purified proteins were immunoblotted using anti–YB-1 antibodies. Biotin end-tagged 5′ UTR of β-Globin was used as a control. (H) Full-length G3BP1 5′ UTR or the M4 deletion mutant (Δ48–171) were transfected into siControl or siYB-1 kd U2OS cells. Lysates were prepared and subjected to RNA affinity chromatography and immunoblotted as described in G to detect 5′ UTR–bound YB-1. Untransfected cells served as controls.

    Techniques Used: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Construct, Luciferase, In Vitro, Recombinant, Activity Assay, Binding Assay, Labeling, Mobility Shift, Concentration Assay, Clone Assay, Affinity Chromatography, Affinity Purification, Mutagenesis, Transfection

    12) Product Images from "LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells"

    Article Title: LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

    Journal: Nature Communications

    doi: 10.1038/ncomms13608

    LncBRM associates with BRM to initiate the BRG1/BRM switch. ( a ) LncBRM intron sequence (Ctrl), lncBRM antisense ( Lnc-AS ) and lncBRM transcripts were labelled with biotin and incubated with oncosphere lysates, followed by silver staining and mass spectrometry. Black arrow denotes BRM. ( b ) RNA pulldown was conducted using lncBRM transcript, followed by immunoblotting. ( c ) Domain mapping of lncBRM transcript. ( d ) LncBRM was incubated with increased doses of BRM, followed by electrophoretic mobility shift assay (EMSA). The 3 segment of lncBRM was labelled with biotin for probing. ( e ) Non-spheres and spheres were visualized by fluorescence in situ hybridization (FISH). Scale bar, 10 μm. ( f ) Antibodies against BRM or BRG1 were used for RNA immunoprecipitation, followed by RT–qPCR. ACTB served as a negative control. ( g ) Spheres (S) and non-sphere cells (N) were lysed and followed by immunoprecipitation with BAF170 and ARID1A antibodies. BRG1 and BRM enrichment was analysed with western blotting. ( h ) Different doses of lncBRM transcripts were incubated with oncosphere lysates and followed by co-immunoprecipitation (co-IP). ( i ) LncBRM -depleted HCC primary spheres were lysed for co-IP as in h . ( j , k ) The indicated oncosphere lysates were fractionated and followed by size fractionation with glycerol gradient ultracentrifugation. Elute gradients were used for western blotting. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis, *** P
    Figure Legend Snippet: LncBRM associates with BRM to initiate the BRG1/BRM switch. ( a ) LncBRM intron sequence (Ctrl), lncBRM antisense ( Lnc-AS ) and lncBRM transcripts were labelled with biotin and incubated with oncosphere lysates, followed by silver staining and mass spectrometry. Black arrow denotes BRM. ( b ) RNA pulldown was conducted using lncBRM transcript, followed by immunoblotting. ( c ) Domain mapping of lncBRM transcript. ( d ) LncBRM was incubated with increased doses of BRM, followed by electrophoretic mobility shift assay (EMSA). The 3 segment of lncBRM was labelled with biotin for probing. ( e ) Non-spheres and spheres were visualized by fluorescence in situ hybridization (FISH). Scale bar, 10 μm. ( f ) Antibodies against BRM or BRG1 were used for RNA immunoprecipitation, followed by RT–qPCR. ACTB served as a negative control. ( g ) Spheres (S) and non-sphere cells (N) were lysed and followed by immunoprecipitation with BAF170 and ARID1A antibodies. BRG1 and BRM enrichment was analysed with western blotting. ( h ) Different doses of lncBRM transcripts were incubated with oncosphere lysates and followed by co-immunoprecipitation (co-IP). ( i ) LncBRM -depleted HCC primary spheres were lysed for co-IP as in h . ( j , k ) The indicated oncosphere lysates were fractionated and followed by size fractionation with glycerol gradient ultracentrifugation. Elute gradients were used for western blotting. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis, *** P

    Techniques Used: Sequencing, Incubation, Silver Staining, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Immunoprecipitation, Quantitative RT-PCR, Negative Control, Western Blot, Co-Immunoprecipitation Assay, Fractionation, Two Tailed Test

    KLF4 binds YAP1 promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression. ( a ) HCC primary spheres were collected for co-IP assays with KLF4 antibody. ( b ) ChIP assay was performed using Klf4 antibody. ( c ) The interaction of KLF4 with YAP1 promoter was verified by EMSA assay. ( d ) HCC primary sphere cells were crosslinked with formaldehyde for ChIP assay with KLF4 antibody, followed by glycerol gradient ultracentrifugation. Elution gradients were concentrated for western blotting (upper panels) and PCR (lower panels) analyses. ( e , f ) KLF4 KO cells were established using CRISPR/Cas9 technology and allowed for sphere formation, followed by ChIP assay using BRG1 ( e ) and H3K4me3 ( f ) antibodies. ( g ) BRG1 was overexpressed in YAP1 promoter mutant ( YAP1p Mut) and wild-type (WT) cells, followed by sphere formation. Oncosphere cells were used for ChIP assays with H3K4me3 and H3K27ac antibodies, followed by examination for YAP1 promoter enrichment with real-time PCR. ( h ) LncBRM or BRG1 was overexpressed in YAP1 promoter mutant and WT cells and collected for RNA extraction. YAP1 mRNA expression was detected with northern blotting. ACTB served as a loading control. Data are shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis; * P
    Figure Legend Snippet: KLF4 binds YAP1 promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression. ( a ) HCC primary spheres were collected for co-IP assays with KLF4 antibody. ( b ) ChIP assay was performed using Klf4 antibody. ( c ) The interaction of KLF4 with YAP1 promoter was verified by EMSA assay. ( d ) HCC primary sphere cells were crosslinked with formaldehyde for ChIP assay with KLF4 antibody, followed by glycerol gradient ultracentrifugation. Elution gradients were concentrated for western blotting (upper panels) and PCR (lower panels) analyses. ( e , f ) KLF4 KO cells were established using CRISPR/Cas9 technology and allowed for sphere formation, followed by ChIP assay using BRG1 ( e ) and H3K4me3 ( f ) antibodies. ( g ) BRG1 was overexpressed in YAP1 promoter mutant ( YAP1p Mut) and wild-type (WT) cells, followed by sphere formation. Oncosphere cells were used for ChIP assays with H3K4me3 and H3K27ac antibodies, followed by examination for YAP1 promoter enrichment with real-time PCR. ( h ) LncBRM or BRG1 was overexpressed in YAP1 promoter mutant and WT cells and collected for RNA extraction. YAP1 mRNA expression was detected with northern blotting. ACTB served as a loading control. Data are shown as means±s.d. Two-tailed Student's t -test was used for statistical analysis; * P

    Techniques Used: Expressing, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Western Blot, Polymerase Chain Reaction, CRISPR, Mutagenesis, Real-time Polymerase Chain Reaction, RNA Extraction, Northern Blot, Two Tailed Test

    BRG1-embedded BAF complex initiates YAP1 signalling in liver CSCs. ( a ) BRG1 and BRM KO cells were established by CRISPR/Cas9 approaches, followed by examination of main self-renewal pathway target genes. Gene fold changes were determined by RT–qPCR. CRISPR/Cas9 caused frameshift mutations with no changes in mRNA levels. ( b ) YAP1 targets were tested by immunoblotting in BRM KO spheres (left panel) or BRG1 KO spheres (right panel). ( c ) Schematic diagram of YAP1 promoter (left panel) and domain mapping (right panel). HCC primary spheres were collected for ChIP assay with BRG1, BRM antibodies. TSS, transcription start site. ( d ) The binding region of YAP1 promoter to BRG1 was validated by luciferase assay. ( e ) Biotin-labelled YAP1 promoter region (−420∼−380 bp) was used for EMSA assay. BRG1 was immunoprecipitated from Huh7 spheres using BRG1-specific antibody. ( f ) Oncosphere nuclear lysates of the indicated cells were treated with DNase I, followed by real-time PCR. ( g ) BRG1 KO or BRM KO spheres were used for ChIP assays using H3K4me3 antibody. ( h ) BRG1-binding region of YAP1 promoter was deleted in HCC primary tumour cells using CRISPR/Cas9 technology, followed by depletion of BRG1 or BRM . Total RNA was extracted for PCR assay. YAP1 PKO, YAP1 promoter KO. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis; * P
    Figure Legend Snippet: BRG1-embedded BAF complex initiates YAP1 signalling in liver CSCs. ( a ) BRG1 and BRM KO cells were established by CRISPR/Cas9 approaches, followed by examination of main self-renewal pathway target genes. Gene fold changes were determined by RT–qPCR. CRISPR/Cas9 caused frameshift mutations with no changes in mRNA levels. ( b ) YAP1 targets were tested by immunoblotting in BRM KO spheres (left panel) or BRG1 KO spheres (right panel). ( c ) Schematic diagram of YAP1 promoter (left panel) and domain mapping (right panel). HCC primary spheres were collected for ChIP assay with BRG1, BRM antibodies. TSS, transcription start site. ( d ) The binding region of YAP1 promoter to BRG1 was validated by luciferase assay. ( e ) Biotin-labelled YAP1 promoter region (−420∼−380 bp) was used for EMSA assay. BRG1 was immunoprecipitated from Huh7 spheres using BRG1-specific antibody. ( f ) Oncosphere nuclear lysates of the indicated cells were treated with DNase I, followed by real-time PCR. ( g ) BRG1 KO or BRM KO spheres were used for ChIP assays using H3K4me3 antibody. ( h ) BRG1-binding region of YAP1 promoter was deleted in HCC primary tumour cells using CRISPR/Cas9 technology, followed by depletion of BRG1 or BRM . Total RNA was extracted for PCR assay. YAP1 PKO, YAP1 promoter KO. Data are shown as means±s.d. Two tailed Student's t -test was used for statistical analysis; * P

    Techniques Used: CRISPR, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Two Tailed Test

    13) Product Images from "LncMAPK6 drives MAPK6 expression and liver TIC self-renewal"

    Article Title: LncMAPK6 drives MAPK6 expression and liver TIC self-renewal

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0770-y

    LncMAPK6 interacted with RNA polymerase II. ( a ) LncMAPK6 was transcribed in vitro for RNA pulldown, and the denoted band in lncMAPK6 sample was identified as POLR2A. ( b ) The binding of lncMAPK6 and POLR2A was examined by Western blot. ( c ) LncMAPK6 truncates were generated (left panels), and incubated with sphere lysates. The interaction between lncMAPK6 truncates and POLR2A was confirmed (right panels). ( d ) RNA EMSA was performed for the combination between lncMAPK6 and POLR2A. The second truncate of lncMAPK6 was used for RNA EMSA. ( e ) Oncospheres derived from clinical samples were used for RNA immunoprecipitation (RIP) assay, and enrichment of lncMAPK6 were detected with realtime PCR. GAPDH was a control. ( f ) The co-localization of lncMAPK6 and POLR2A was confirmed by double FISH assay. Scale bars, 10 μm. ( g , h ) LncMAPK6 depleted ( g ) and overexpressed ( h ) cells were crushed for POLR2A ChIP, followed by detection for MAPK6 promoter enrichment with realtime PCR
    Figure Legend Snippet: LncMAPK6 interacted with RNA polymerase II. ( a ) LncMAPK6 was transcribed in vitro for RNA pulldown, and the denoted band in lncMAPK6 sample was identified as POLR2A. ( b ) The binding of lncMAPK6 and POLR2A was examined by Western blot. ( c ) LncMAPK6 truncates were generated (left panels), and incubated with sphere lysates. The interaction between lncMAPK6 truncates and POLR2A was confirmed (right panels). ( d ) RNA EMSA was performed for the combination between lncMAPK6 and POLR2A. The second truncate of lncMAPK6 was used for RNA EMSA. ( e ) Oncospheres derived from clinical samples were used for RNA immunoprecipitation (RIP) assay, and enrichment of lncMAPK6 were detected with realtime PCR. GAPDH was a control. ( f ) The co-localization of lncMAPK6 and POLR2A was confirmed by double FISH assay. Scale bars, 10 μm. ( g , h ) LncMAPK6 depleted ( g ) and overexpressed ( h ) cells were crushed for POLR2A ChIP, followed by detection for MAPK6 promoter enrichment with realtime PCR

    Techniques Used: In Vitro, Binding Assay, Western Blot, Generated, Incubation, Derivative Assay, Immunoprecipitation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation

    14) Product Images from "Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii"

    Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-7-141

    RNA binding analysis of rTgPuf1 PUM-HD. EMSA titration of rTgPuf1 PUM-HD binding toPfs28 RNA1 (A) , hb NRE RNA (B) , and Pfs28 RNA1M (C) . In Pfs28 RNA1M, the UGU sequence in the putative PBE of Pfs28 RNA1 was mutated to UCC. (D) , (E) and (F) Quantitation of dissociation constant (Kd) values based on EMSA analysis from (A) , (B) and (C) , respectively.
    Figure Legend Snippet: RNA binding analysis of rTgPuf1 PUM-HD. EMSA titration of rTgPuf1 PUM-HD binding toPfs28 RNA1 (A) , hb NRE RNA (B) , and Pfs28 RNA1M (C) . In Pfs28 RNA1M, the UGU sequence in the putative PBE of Pfs28 RNA1 was mutated to UCC. (D) , (E) and (F) Quantitation of dissociation constant (Kd) values based on EMSA analysis from (A) , (B) and (C) , respectively.

    Techniques Used: RNA Binding Assay, Titration, Binding Assay, Sequencing, Quantitation Assay

    TgPuf1 binds conserved RNA motifs. Competition EMSA shows the specificities of rTgPuf1 binding to Pfs28 RNA1 but not to mutant Pfs28 RNA1. Competitor RNAs were added in reactions at 5X, 50X and 100X of the biotinylated probe (labeled with an asterisk).
    Figure Legend Snippet: TgPuf1 binds conserved RNA motifs. Competition EMSA shows the specificities of rTgPuf1 binding to Pfs28 RNA1 but not to mutant Pfs28 RNA1. Competitor RNAs were added in reactions at 5X, 50X and 100X of the biotinylated probe (labeled with an asterisk).

    Techniques Used: Binding Assay, Mutagenesis, Labeling

    15) Product Images from "LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3"

    Article Title: LncKdm2b controls self‐renewal of embryonic stem cells via activating expression of transcription factor Zbtb3

    Journal: The EMBO Journal

    doi: 10.15252/embj.201797174

    LncKdm2b promotes the ATPase activity of SRCAP A Biotin RNA pull‐downs were performed with nuclear extracts of mouse ESCs using full‐length lncKdm2b transcript (Sense), antisense, and Xist A repeats sequence control followed with mass spectrometry. B, C The interaction of SRCAP with lncKdm2b was confirmed by immunoblotting (B) and CHIRP assay (C). Biotinylated probes were hybridized to lncKdm2b . D Pluripotent ESCs and E3.5 blastocysts were probed with lncKdm2b by RNA‐FISH, followed by immunofluorescence staining for SRCAP. Green: lncKdm2b probe; red: SRCAP; nuclei were counterstained by DAPI. Scale bar, 10 μm. For normal ESC clone, n = 240; for LIF‐withdrawal ESC clone, n = 130; for lncKdm2b −/− ESC clone, n = 105; for E3.5 embryos, n = 119. E Interaction of lncKdm2b with SRCAP was verified by RIP assay. ESC lysates were incubated with anti‐SRCAP antibody, followed by RNA immunoprecipitation (RIP) assay. RNA was extracted and reversely transcribed. LncKdm2b . F Full‐length and truncated fragments of lncKdm2b were in vitro ‐transcribed to biotin‐labeled RNA followed with RNA pull‐down and immunoblotting. d450–700 denotes the truncated fragment of lncKdm2b deleting nt 450–700. d700–1,000 denotes the truncated fragment of lncKdm2b deleting nt 700–1,000. G Nuclear extracts of ESCs and biotin‐labeled lncKdm2b (450–700 nt) probes were incubated for EMSA assays. Anti‐SRCAP antibody was preincubated with nuclear extracts that caused supershift. H ESC lysates were immunoprecipitated with anti‐SRCAP antibody, followed by detection of ATPase activities. Biotin‐labeled lncKdm2b and lncKdm2b (nt 450–700) fragments were generated by in vitro transcription by T7 RNA polymerase. Mouse IgG IP was used as a background control. Relative OD values were normalized to IgG background control and shown as fold changes as means ± SD. lnc, lncKdm2b ; oe, overexpression. ** P = 0.0034, ** P = 0.0089, ** P = 0.0023, ** P = 0.0011 by unpaired Student's t ‐test. I lncKdm2b +/+ and lncKdm2b −/− ESC cell lysates were immunoprecipitated with anti‐SRCAP antibody, followed by immunoblotting with the indicated antibodies. Data information: All data are representative of five independent experiments.
    Figure Legend Snippet: LncKdm2b promotes the ATPase activity of SRCAP A Biotin RNA pull‐downs were performed with nuclear extracts of mouse ESCs using full‐length lncKdm2b transcript (Sense), antisense, and Xist A repeats sequence control followed with mass spectrometry. B, C The interaction of SRCAP with lncKdm2b was confirmed by immunoblotting (B) and CHIRP assay (C). Biotinylated probes were hybridized to lncKdm2b . D Pluripotent ESCs and E3.5 blastocysts were probed with lncKdm2b by RNA‐FISH, followed by immunofluorescence staining for SRCAP. Green: lncKdm2b probe; red: SRCAP; nuclei were counterstained by DAPI. Scale bar, 10 μm. For normal ESC clone, n = 240; for LIF‐withdrawal ESC clone, n = 130; for lncKdm2b −/− ESC clone, n = 105; for E3.5 embryos, n = 119. E Interaction of lncKdm2b with SRCAP was verified by RIP assay. ESC lysates were incubated with anti‐SRCAP antibody, followed by RNA immunoprecipitation (RIP) assay. RNA was extracted and reversely transcribed. LncKdm2b . F Full‐length and truncated fragments of lncKdm2b were in vitro ‐transcribed to biotin‐labeled RNA followed with RNA pull‐down and immunoblotting. d450–700 denotes the truncated fragment of lncKdm2b deleting nt 450–700. d700–1,000 denotes the truncated fragment of lncKdm2b deleting nt 700–1,000. G Nuclear extracts of ESCs and biotin‐labeled lncKdm2b (450–700 nt) probes were incubated for EMSA assays. Anti‐SRCAP antibody was preincubated with nuclear extracts that caused supershift. H ESC lysates were immunoprecipitated with anti‐SRCAP antibody, followed by detection of ATPase activities. Biotin‐labeled lncKdm2b and lncKdm2b (nt 450–700) fragments were generated by in vitro transcription by T7 RNA polymerase. Mouse IgG IP was used as a background control. Relative OD values were normalized to IgG background control and shown as fold changes as means ± SD. lnc, lncKdm2b ; oe, overexpression. ** P = 0.0034, ** P = 0.0089, ** P = 0.0023, ** P = 0.0011 by unpaired Student's t ‐test. I lncKdm2b +/+ and lncKdm2b −/− ESC cell lysates were immunoprecipitated with anti‐SRCAP antibody, followed by immunoblotting with the indicated antibodies. Data information: All data are representative of five independent experiments.

    Techniques Used: Activity Assay, Sequencing, Mass Spectrometry, Fluorescence In Situ Hybridization, Immunofluorescence, Staining, Incubation, Immunoprecipitation, In Vitro, Labeling, Generated, Over Expression

    16) Product Images from "LncMAPK6 drives MAPK6 expression and liver TIC self-renewal"

    Article Title: LncMAPK6 drives MAPK6 expression and liver TIC self-renewal

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-018-0770-y

    LncMAPK6 interacted with RNA polymerase II. ( a ) LncMAPK6 was transcribed in vitro for RNA pulldown, and the denoted band in lncMAPK6 sample was identified as POLR2A. ( b ) The binding of lncMAPK6 and POLR2A was examined by Western blot. ( c ) LncMAPK6 truncates were generated (left panels), and incubated with sphere lysates. The interaction between lncMAPK6 truncates and POLR2A was confirmed (right panels). ( d ) RNA EMSA was performed for the combination between lncMAPK6 and POLR2A. The second truncate of lncMAPK6 was used for RNA EMSA. ( e ) Oncospheres derived from clinical samples were used for RNA immunoprecipitation (RIP) assay, and enrichment of lncMAPK6 were detected with realtime PCR. GAPDH was a control. ( f ) The co-localization of lncMAPK6 and POLR2A was confirmed by double FISH assay. Scale bars, 10 μm. ( g , h ) LncMAPK6 depleted ( g ) and overexpressed ( h ) cells were crushed for POLR2A ChIP, followed by detection for MAPK6 promoter enrichment with realtime PCR
    Figure Legend Snippet: LncMAPK6 interacted with RNA polymerase II. ( a ) LncMAPK6 was transcribed in vitro for RNA pulldown, and the denoted band in lncMAPK6 sample was identified as POLR2A. ( b ) The binding of lncMAPK6 and POLR2A was examined by Western blot. ( c ) LncMAPK6 truncates were generated (left panels), and incubated with sphere lysates. The interaction between lncMAPK6 truncates and POLR2A was confirmed (right panels). ( d ) RNA EMSA was performed for the combination between lncMAPK6 and POLR2A. The second truncate of lncMAPK6 was used for RNA EMSA. ( e ) Oncospheres derived from clinical samples were used for RNA immunoprecipitation (RIP) assay, and enrichment of lncMAPK6 were detected with realtime PCR. GAPDH was a control. ( f ) The co-localization of lncMAPK6 and POLR2A was confirmed by double FISH assay. Scale bars, 10 μm. ( g , h ) LncMAPK6 depleted ( g ) and overexpressed ( h ) cells were crushed for POLR2A ChIP, followed by detection for MAPK6 promoter enrichment with realtime PCR

    Techniques Used: In Vitro, Binding Assay, Western Blot, Generated, Incubation, Derivative Assay, Immunoprecipitation, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation

    17) Product Images from "PLF‐1 (Proliferin‐1) Modulates Smooth Muscle Cell Proliferation and Development of Experimental Intimal Hyperplasia"

    Article Title: PLF‐1 (Proliferin‐1) Modulates Smooth Muscle Cell Proliferation and Development of Experimental Intimal Hyperplasia

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.117.005886

    PLF ‐1 activates cell cycle. Mouse SMC s were pla ted in 6‐well plates (2.5×10 5 /well), allowed to adhere overnight, and then incubated in the presence or absence of rPLF ‐1 at 20 and 100 ng/mL for 24 hours. After trypsinization and centrifugation, cell pellets were suspended and fixed in 70% ethanol at 4°C overnight and then evaluated for propidium iodide labeling of DNA by flow cytometry. Representative histograms of DNA content during the cell cycle ( A through C ). Distribution of cells in G2/S/M expressed as a percentage of total cells (n=7; D ). PLF‐1 indicates proliferin‐1; rPLF ‐1, recombinant proliferin‐1; SMCs, smooth muscle cells.
    Figure Legend Snippet: PLF ‐1 activates cell cycle. Mouse SMC s were pla ted in 6‐well plates (2.5×10 5 /well), allowed to adhere overnight, and then incubated in the presence or absence of rPLF ‐1 at 20 and 100 ng/mL for 24 hours. After trypsinization and centrifugation, cell pellets were suspended and fixed in 70% ethanol at 4°C overnight and then evaluated for propidium iodide labeling of DNA by flow cytometry. Representative histograms of DNA content during the cell cycle ( A through C ). Distribution of cells in G2/S/M expressed as a percentage of total cells (n=7; D ). PLF‐1 indicates proliferin‐1; rPLF ‐1, recombinant proliferin‐1; SMCs, smooth muscle cells.

    Techniques Used: Proximity Ligation Assay, Incubation, Centrifugation, Labeling, Flow Cytometry, Cytometry, Recombinant

    18) Product Images from "Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1"

    Article Title: Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1

    Journal: Nature Communications

    doi: 10.1038/ncomms8318

    MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.
    Figure Legend Snippet: MiR-27b regulates the resistance of breast cancer cells to docetaxel. ( a ) Overview of the method used to establish miR-27b knockdown MCF7-luc (MCF7-luc anti-miR-27b) cells. ( b , c ) Dose–response curves of MCF7-luc anti-NC, MCF7-luc anti-miR-27b and MCF7-luc miR-27b o.e. cells treated with docetaxel. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( d ) Morphologies of the MCF7-luc anti-NC, MCF7-luc miR-27b o.e. and MCF7-luc anti-miR-27b cells. Scale bar, 100 μm. ( e ) Flow cytometric analyses of the SP fraction of MCF7-luc derivatives in the presence and absence of Ko143. ( f ) Quantification of the SP fraction of MCF7-luc derivatives. The SP fraction was determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. Statistical significance was determined by Student's t -test.

    Techniques Used: Flow Cytometry, Staining

    Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.
    Figure Legend Snippet: Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

    Techniques Used: Functional Assay, Flow Cytometry, Staining, Transfection, shRNA, Proximity Ligation Assay, Expressing, In Vitro, Binding Assay, Purification

    19) Product Images from "Sensitive and specific miRNA detection method using SplintR Ligase"

    Article Title: Sensitive and specific miRNA detection method using SplintR Ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw399

    Comparison of SplintR ligation method to TaqMan assay for detection of miR-122 in rat liver RNA. Three different concentrations of rat liver total RNA were used; 1 ng (green), 0.2 ng (blue), 0.04 ng/μ (red) and no rat liver RNA (black). ( A ) This panel shows the qPCR traces for the SplintR method described in Figure 2 . The ligated probes were amplified by PCR and detected with a double quenched miR-122 specific DNA probe. ( B ) TaqMan detection method for miR-122 used a DNA hairpin complementary to the 3′ end of the miRNA to prime cDNA synthesis with reverse transcriptase. The amplified product is detected by cleavage of a TaqMan probe specific for miR-122 (miR-122 sqp). The sequences of the probes and protocols for amplification and detection are described in Materials and Methods.
    Figure Legend Snippet: Comparison of SplintR ligation method to TaqMan assay for detection of miR-122 in rat liver RNA. Three different concentrations of rat liver total RNA were used; 1 ng (green), 0.2 ng (blue), 0.04 ng/μ (red) and no rat liver RNA (black). ( A ) This panel shows the qPCR traces for the SplintR method described in Figure 2 . The ligated probes were amplified by PCR and detected with a double quenched miR-122 specific DNA probe. ( B ) TaqMan detection method for miR-122 used a DNA hairpin complementary to the 3′ end of the miRNA to prime cDNA synthesis with reverse transcriptase. The amplified product is detected by cleavage of a TaqMan probe specific for miR-122 (miR-122 sqp). The sequences of the probes and protocols for amplification and detection are described in Materials and Methods.

    Techniques Used: Ligation, TaqMan Assay, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    20) Product Images from "A robust gene-stacking method utilizing yeast assembly for plant synthetic biology"

    Article Title: A robust gene-stacking method utilizing yeast assembly for plant synthetic biology

    Journal: Nature Communications

    doi: 10.1038/ncomms13215

    jStack hierarchical assembly. ( a ) A library of Level 0 parts composed of linkers (linker, L), promoters (Prom., P), coding sequences (CDS; C) and terminators (Term., T) are available. Level 1 assembly consists of digesting desired Level 0 parts along with their intermediate vector backbone (containing a complementary antibiotic selection; chloramphenicol, Cm) with a Type IIS restriction enzyme followed by ligation facilitated by compatible sticky ends. Type IIS enzymes generating compatible sticky ends are denoted by the tiny black arrows above Level 0 plasmids. A GFP dropout-cassette is cut out from the intermediate vector backbone allowing for green/white selection, where all the white colonies have correctly assembled Level 1 constructs (illustrated in top right grey box). Compatibility of liberated ends by the Type IIS restriction enzymes (illustrated with coloured box at the end of each DNA part) allows assembling of all DNA parts in the correct order. ( b ) Various Level 1 constructs with compatible linker and terminator sequences that have homology to one another are digested with one of three flanking rare Type II restriction enzyme cutsites (NotI, SbfI or AscI). These are all transformed into yeast with a PmlI linearized pYB vector, which loses the Ura3 dropout-cassette. Homology between linker and terminator sequences allows DNA assembly via in vivo yeast recombination in the correct order, circularizing the pYB vector. The loss of the Ura3 cassette and the presence of a Leu2 marker on the backbone confer the resistance to 5-FOA and autotrophy for leucine, respectively; thus, any colonies that grow on minimal SD-Leu supplemented with 5-FOA, have recombined the various DNA fragments together into the pYB vector.
    Figure Legend Snippet: jStack hierarchical assembly. ( a ) A library of Level 0 parts composed of linkers (linker, L), promoters (Prom., P), coding sequences (CDS; C) and terminators (Term., T) are available. Level 1 assembly consists of digesting desired Level 0 parts along with their intermediate vector backbone (containing a complementary antibiotic selection; chloramphenicol, Cm) with a Type IIS restriction enzyme followed by ligation facilitated by compatible sticky ends. Type IIS enzymes generating compatible sticky ends are denoted by the tiny black arrows above Level 0 plasmids. A GFP dropout-cassette is cut out from the intermediate vector backbone allowing for green/white selection, where all the white colonies have correctly assembled Level 1 constructs (illustrated in top right grey box). Compatibility of liberated ends by the Type IIS restriction enzymes (illustrated with coloured box at the end of each DNA part) allows assembling of all DNA parts in the correct order. ( b ) Various Level 1 constructs with compatible linker and terminator sequences that have homology to one another are digested with one of three flanking rare Type II restriction enzyme cutsites (NotI, SbfI or AscI). These are all transformed into yeast with a PmlI linearized pYB vector, which loses the Ura3 dropout-cassette. Homology between linker and terminator sequences allows DNA assembly via in vivo yeast recombination in the correct order, circularizing the pYB vector. The loss of the Ura3 cassette and the presence of a Leu2 marker on the backbone confer the resistance to 5-FOA and autotrophy for leucine, respectively; thus, any colonies that grow on minimal SD-Leu supplemented with 5-FOA, have recombined the various DNA fragments together into the pYB vector.

    Techniques Used: Plasmid Preparation, Selection, Ligation, Construct, Transformation Assay, In Vivo, Marker

    21) Product Images from "An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation"

    Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006500

    EBNA3C regulates Bcl6 mRNA expression through inhibition of its promoter activity. A) 5 million BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were harvested and extracted total RNA using Trizol reagent. Then cDNA was prepared with reverse transcriptase kit, and detected Bcl6 mRNA expression by quantitative Real-time PCR analysis (SYBR green). GAPDH was set as an internal reference. Each sample was determined in triplicate. B) EBNA3C knock-down (sh-E3C) stable LCL1 or control (sh-Ctrl) LCL1 cells were harvested and Bcl6 mRNA expression was detected using Real-time PCR as mentioned. C) 10 million BJAB10 cells were transfected with specific EBNA3C (sh-E3C) or control (sh-Ctrl) short hairpin RNA. At 48 hours post-transfection, total RNA was extracted, reverse-transcribed, followed by quantitative Real-time PCR analysis. Meanwhile, EBNA3C expression was also detected by western blot analysis. D) HEK293T cells were transfected with the reporter constructs containing wild-type Bcl6 promoter (pLA/B9) and increasing amount of Myc-EBNA3C. Cells were collected and lysed in lysis buffer at 48 hours post-transfection. Luciferase activity was measured according to the dual-luciferase reporter assay kit. Mean values and standard deviations of two independent experiments were presented. Cell lysate was resolved by 10% SDS-PAGE in order to check EBNA3C expression. GAPDH western blot was done as an internal loading control. E) HEK293T cells were transfected with wild-type Bcl6 promoter reporter plasmids in combination with different expression constructs as indicated. Cells were collected and lysed, then the lysate were used to detect luciferase activity as previously described.
    Figure Legend Snippet: EBNA3C regulates Bcl6 mRNA expression through inhibition of its promoter activity. A) 5 million BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were harvested and extracted total RNA using Trizol reagent. Then cDNA was prepared with reverse transcriptase kit, and detected Bcl6 mRNA expression by quantitative Real-time PCR analysis (SYBR green). GAPDH was set as an internal reference. Each sample was determined in triplicate. B) EBNA3C knock-down (sh-E3C) stable LCL1 or control (sh-Ctrl) LCL1 cells were harvested and Bcl6 mRNA expression was detected using Real-time PCR as mentioned. C) 10 million BJAB10 cells were transfected with specific EBNA3C (sh-E3C) or control (sh-Ctrl) short hairpin RNA. At 48 hours post-transfection, total RNA was extracted, reverse-transcribed, followed by quantitative Real-time PCR analysis. Meanwhile, EBNA3C expression was also detected by western blot analysis. D) HEK293T cells were transfected with the reporter constructs containing wild-type Bcl6 promoter (pLA/B9) and increasing amount of Myc-EBNA3C. Cells were collected and lysed in lysis buffer at 48 hours post-transfection. Luciferase activity was measured according to the dual-luciferase reporter assay kit. Mean values and standard deviations of two independent experiments were presented. Cell lysate was resolved by 10% SDS-PAGE in order to check EBNA3C expression. GAPDH western blot was done as an internal loading control. E) HEK293T cells were transfected with wild-type Bcl6 promoter reporter plasmids in combination with different expression constructs as indicated. Cells were collected and lysed, then the lysate were used to detect luciferase activity as previously described.

    Techniques Used: Expressing, Inhibition, Activity Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transfection, shRNA, Western Blot, Construct, Proximity Ligation Assay, Lysis, Luciferase, Reporter Assay, SDS Page

    22) Product Images from "Intracellular S100A9 Promotes Myeloid-Derived Suppressor Cells during Late Sepsis"

    Article Title: Intracellular S100A9 Promotes Myeloid-Derived Suppressor Cells during Late Sepsis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01565

    The S100A8 and S100A9 proteins are retained in Gr1 + CD11b + cells during late sepsis. Gr1 + CD11b + cells were isolated from the bone marrow cells by positive selection. The early and late sepsis groups, respectively, included mice that were killed between days 1–5 and 6–28 after cecal ligation and puncture. (A) Levels of S100A8 and S100A9 mRNAs. Total RNA was extracted from Gr1 + CD11b + cells, and mRNA levels were determined by real-time PCR. The S100A8 and S100A9 expression levels were normalized to 18S rRNA (* p
    Figure Legend Snippet: The S100A8 and S100A9 proteins are retained in Gr1 + CD11b + cells during late sepsis. Gr1 + CD11b + cells were isolated from the bone marrow cells by positive selection. The early and late sepsis groups, respectively, included mice that were killed between days 1–5 and 6–28 after cecal ligation and puncture. (A) Levels of S100A8 and S100A9 mRNAs. Total RNA was extracted from Gr1 + CD11b + cells, and mRNA levels were determined by real-time PCR. The S100A8 and S100A9 expression levels were normalized to 18S rRNA (* p

    Techniques Used: Isolation, Selection, Mouse Assay, Ligation, Real-time Polymerase Chain Reaction, Expressing

    23) Product Images from "Mapping three-dimensional genome architecture through in situ DNase Hi-C"

    Article Title: Mapping three-dimensional genome architecture through in situ DNase Hi-C

    Journal: Nature protocols

    doi: 10.1038/nprot.2016.126

    Digestion quality controls throughout the  in situ  DNase Hi-C protocol a.) A typical digestion pattern for DNase I-digested fixed chromatin prior to proximity ligation, run on a 6% TBE-PAGE gel. b.) Example of the BamH1 quality control experiment performed on GM12878  in situ  DNase Hi-C libraries; in this example, BamH1 shifts the  in situ  DNase Hi-C library by digesting the reconstituted BamH1 site that forms following proximity ligation of the biotinylated bridge adaptors. Crucially, digestion with another 6-cutter (EcoR1), does not recapitulate this pattern, proving that the BamH1 digestion is specific to proximity ligated fragments. All reactions were run on one 6% TBE-PAGE gel.
    Figure Legend Snippet: Digestion quality controls throughout the in situ DNase Hi-C protocol a.) A typical digestion pattern for DNase I-digested fixed chromatin prior to proximity ligation, run on a 6% TBE-PAGE gel. b.) Example of the BamH1 quality control experiment performed on GM12878 in situ DNase Hi-C libraries; in this example, BamH1 shifts the in situ DNase Hi-C library by digesting the reconstituted BamH1 site that forms following proximity ligation of the biotinylated bridge adaptors. Crucially, digestion with another 6-cutter (EcoR1), does not recapitulate this pattern, proving that the BamH1 digestion is specific to proximity ligated fragments. All reactions were run on one 6% TBE-PAGE gel.

    Techniques Used: In Situ, Hi-C, Ligation, Polyacrylamide Gel Electrophoresis

    24) Product Images from "Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure"

    Article Title: Actual Ligation Frequencies in the Chromosome Conformation Capture Procedure

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060403

    The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).
    Figure Legend Snippet: The kinetics of the ligation reaction in the presence or absence of SDS. Electrophoretic separation of the products obtained upon the ligation of pUC18 Hind III fragments for the indicated times in the presence or absence of SDS and Triton X-100 in an ethidium bromide-stained agarose gel. The reaction was carried out in 1× T4 DNA Ligase Buffer (Fermentas) with 100 ng/ µl DNA and 0.1 U/ µl T4 DNA ligase (Fermentas). M–DNA size marker (Fermentas, SM0331).

    Techniques Used: Ligation, Staining, Agarose Gel Electrophoresis, Marker

    25) Product Images from "Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine"

    Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm657

    ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.
    Figure Legend Snippet: ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.

    Techniques Used: Modification, Ligation

    26) Product Images from "Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements"

    Article Title: Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements

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

    doi: 10.1073/pnas.0406281101

    Properties of Penelope -encoded EN and its mutant derivatives. ( A and B ) Cleavage and DNA-binding assays. The 38-bp ps1 target ( A Upper ) was labeled with [α- 32 P]dATP at both ends (in bold italics). Lane C, control without protein. The remaining seven lanes in A and B represent cleavage and DNA-binding assays, respectively, for the EN protein and its mutant variants: Y730H (2%); Y746H (0%); R757A (100%); H781G (27%); D792A (5%); E800G (0%); and WT, wild-type Penelope EN (100%); numbers in parentheses designate the relative cleavage activity with respect to WT obtained from PhosphorImager quantitative scans of gel A . To suppress target cleavage, binding reactions in B were performed without Mg 2+ and in the presence of EDTA; however, residual cleavage is still visible for the most active R757A and WT proteins. ( C ) Binding of WT Penelope EN to the ps1 target at different concentrations of poly(dI)·poly(dC). Lane C, control without protein; WT, wild-type Penelope EN in a standard binding reaction with 0.1 mg/ml BSA. In the remaining lanes, increasing amounts of poly(dI)·poly(dC) (from 0.025 to 1 μg) were added to the binding reaction, as indicated. ( D ) Determination of cleavage sites of Penelope EN. The 34-bp fragment of ps1 was used as a target, with the top and bottom strand labeled as indicated. Lane L, 1-bp DNA ladder; WT, labeled fragments digested by WT EN; Y746H, labeled fragments digested by the EN mutant. Target site duplication (TSD) is shown by brackets, and the most prominent cleavage products, which become visible after a 1-h incubation, are shown in bold italics. ( E ) Ligation of the labeled ps1 fragment after digestion by the WT Penelope EN. Lane C, labeled fragment without protein; WT, labeled fragment digested with EN. The remaining lanes show ligation of the cleavage products after 3-, 10-, and 30-min incubation with T4 DNA ligase.
    Figure Legend Snippet: Properties of Penelope -encoded EN and its mutant derivatives. ( A and B ) Cleavage and DNA-binding assays. The 38-bp ps1 target ( A Upper ) was labeled with [α- 32 P]dATP at both ends (in bold italics). Lane C, control without protein. The remaining seven lanes in A and B represent cleavage and DNA-binding assays, respectively, for the EN protein and its mutant variants: Y730H (2%); Y746H (0%); R757A (100%); H781G (27%); D792A (5%); E800G (0%); and WT, wild-type Penelope EN (100%); numbers in parentheses designate the relative cleavage activity with respect to WT obtained from PhosphorImager quantitative scans of gel A . To suppress target cleavage, binding reactions in B were performed without Mg 2+ and in the presence of EDTA; however, residual cleavage is still visible for the most active R757A and WT proteins. ( C ) Binding of WT Penelope EN to the ps1 target at different concentrations of poly(dI)·poly(dC). Lane C, control without protein; WT, wild-type Penelope EN in a standard binding reaction with 0.1 mg/ml BSA. In the remaining lanes, increasing amounts of poly(dI)·poly(dC) (from 0.025 to 1 μg) were added to the binding reaction, as indicated. ( D ) Determination of cleavage sites of Penelope EN. The 34-bp fragment of ps1 was used as a target, with the top and bottom strand labeled as indicated. Lane L, 1-bp DNA ladder; WT, labeled fragments digested by WT EN; Y746H, labeled fragments digested by the EN mutant. Target site duplication (TSD) is shown by brackets, and the most prominent cleavage products, which become visible after a 1-h incubation, are shown in bold italics. ( E ) Ligation of the labeled ps1 fragment after digestion by the WT Penelope EN. Lane C, labeled fragment without protein; WT, labeled fragment digested with EN. The remaining lanes show ligation of the cleavage products after 3-, 10-, and 30-min incubation with T4 DNA ligase.

    Techniques Used: Mutagenesis, Binding Assay, Labeling, Activity Assay, Incubation, Ligation

    27) Product Images from "Genomic Methods for Clinical and Translational Pain Research"

    Article Title: Genomic Methods for Clinical and Translational Pain Research

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-561-9_2

    Recommended workflow for Affymetrix Genome-Wide Human SNP 6.0 Array.
    Figure Legend Snippet: Recommended workflow for Affymetrix Genome-Wide Human SNP 6.0 Array.

    Techniques Used: Genome Wide

    28) Product Images from "Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment"

    Article Title: Autophagy promotes apoptosis of mesenchymal stem cells under inflammatory microenvironment

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-015-0245-4

    Inhibition of autophagy moderately suppresses tumor necrosis factor alpha ( TNF-α ) plus interferon gamma ( IFN-γ )-induced apoptosis of MSCs. a Levels of cytokines in sera of naive mice (n = 8 mice per group) and cecal ligation and puncture ( CLP ) mice (n = 10 mice per group) were determined by ELISA. Data are shown as mean ± SEM. b Naive or CLP mice were treated with phosphate-buffered saline ( PBS ; n = 6 mice per group), shNC-MSCs ( shNC ; n = 6 mice per group) or shBec1-MSCs ( shBecn1 ; n = 6 mice per group) intravenously (1 × 10 6 cells/mouse) for 8 hours after the surgery. Levels of cytokines in sera of naive mice and CLP mice were determined by ELISA. Data are shown as mean ± SEM. c shNC-MSCs and shBec1-MSCs were stimulated with TNF-α (20 ng/ml) plus IFN-γ (50 ng/ml) or starved with EBSS for 24 hours, and then the cells were harvested and stained with annexin V/prodium iodide ( PI ). d Percentage of MSC apoptosis after treatment with TNF-α plus IFN-γ; data are shown as mean ± SEM of five independent experiments. ** P
    Figure Legend Snippet: Inhibition of autophagy moderately suppresses tumor necrosis factor alpha ( TNF-α ) plus interferon gamma ( IFN-γ )-induced apoptosis of MSCs. a Levels of cytokines in sera of naive mice (n = 8 mice per group) and cecal ligation and puncture ( CLP ) mice (n = 10 mice per group) were determined by ELISA. Data are shown as mean ± SEM. b Naive or CLP mice were treated with phosphate-buffered saline ( PBS ; n = 6 mice per group), shNC-MSCs ( shNC ; n = 6 mice per group) or shBec1-MSCs ( shBecn1 ; n = 6 mice per group) intravenously (1 × 10 6 cells/mouse) for 8 hours after the surgery. Levels of cytokines in sera of naive mice and CLP mice were determined by ELISA. Data are shown as mean ± SEM. c shNC-MSCs and shBec1-MSCs were stimulated with TNF-α (20 ng/ml) plus IFN-γ (50 ng/ml) or starved with EBSS for 24 hours, and then the cells were harvested and stained with annexin V/prodium iodide ( PI ). d Percentage of MSC apoptosis after treatment with TNF-α plus IFN-γ; data are shown as mean ± SEM of five independent experiments. ** P

    Techniques Used: Inhibition, Mouse Assay, Ligation, Enzyme-linked Immunosorbent Assay, Staining

    Inhibition of the ROS/ERK pathway is responsible for autophagy-mediated apoptosis of MSCs. a shNC-MSCs ( shNC ) and shBec1-MSCs ( shBecn1 ) stimulated with tumor necrosis factor alpha ( TNF-α ; 20 ng/ml) plus interferon gamma ( IFN-γ ; 50 ng/ml) or combined with PD98059 or N-acetyl cysteine ( NAC ) before stimulation with TNF-α for 24 hours. b Naive MSCs were treated with 3-methyladenine ( 3-Ma ; 10 mM) for 12 hours, then stimulated with TNF-α (50 ng/ml) plus IFN-γ (50 ng/ml) or combined with PD98059 or NAC before TNF-α stimulation for 24 hours. Cells were stained with annexin V/prodium iodide ( PI ) for apoptosis assay. Representative graphs from three independent experiments are shown
    Figure Legend Snippet: Inhibition of the ROS/ERK pathway is responsible for autophagy-mediated apoptosis of MSCs. a shNC-MSCs ( shNC ) and shBec1-MSCs ( shBecn1 ) stimulated with tumor necrosis factor alpha ( TNF-α ; 20 ng/ml) plus interferon gamma ( IFN-γ ; 50 ng/ml) or combined with PD98059 or N-acetyl cysteine ( NAC ) before stimulation with TNF-α for 24 hours. b Naive MSCs were treated with 3-methyladenine ( 3-Ma ; 10 mM) for 12 hours, then stimulated with TNF-α (50 ng/ml) plus IFN-γ (50 ng/ml) or combined with PD98059 or NAC before TNF-α stimulation for 24 hours. Cells were stained with annexin V/prodium iodide ( PI ) for apoptosis assay. Representative graphs from three independent experiments are shown

    Techniques Used: Inhibition, Staining, Apoptosis Assay

    Inhibition of autophagy improves survival of MSCs in CLP mice. a MSCs were stably infected with negative control lentiviral vector ( shNC ) or with vector expressing shRNA to inhibit Beclin-1 ( shBecn1 ); western blot analysis of shNC-MSCs and shBec1-MSCs. b Survival curves of CLP mice treated with phosphate-buffered saline ( PBS ; n = 12 mice per group), shNC-MSCs (n = 14 mice per group) or shBec1-MSCs (n = 13 mice per group) intravenously (1 × 10 6 cells/mouse). c , d shNC-MSCs or shBec1-MSCs were mixed with matrigel and were injected into naive and CLP mice subcutaneously for 24 hours. c Then MSCs were isolated and stained with annexin V/prodium iodide ( PI ) for apoptosis assay. d Percentage of MSC apoptosis after injection into naive (n = 4 mice per group) and CLP mice (n = 4 mice per group); data are shown as mean ± SEM. * P
    Figure Legend Snippet: Inhibition of autophagy improves survival of MSCs in CLP mice. a MSCs were stably infected with negative control lentiviral vector ( shNC ) or with vector expressing shRNA to inhibit Beclin-1 ( shBecn1 ); western blot analysis of shNC-MSCs and shBec1-MSCs. b Survival curves of CLP mice treated with phosphate-buffered saline ( PBS ; n = 12 mice per group), shNC-MSCs (n = 14 mice per group) or shBec1-MSCs (n = 13 mice per group) intravenously (1 × 10 6 cells/mouse). c , d shNC-MSCs or shBec1-MSCs were mixed with matrigel and were injected into naive and CLP mice subcutaneously for 24 hours. c Then MSCs were isolated and stained with annexin V/prodium iodide ( PI ) for apoptosis assay. d Percentage of MSC apoptosis after injection into naive (n = 4 mice per group) and CLP mice (n = 4 mice per group); data are shown as mean ± SEM. * P

    Techniques Used: Inhibition, Mouse Assay, Stable Transfection, Infection, Negative Control, Plasmid Preparation, Expressing, shRNA, Western Blot, Injection, Isolation, Staining, Apoptosis Assay

    29) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:
    Figure Legend Snippet: Low frequency minor alleles are detected at significantly higher frequencies by Illumina MiSeq compared to Ion Torrent PGM. Nucleotide variants were subdivided in two frequency classes: high (frequency minor allele > 15%, n = 4) and low (frequency minor allele:

    Techniques Used:

    Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).
    Figure Legend Snippet: Sequence coverage of the influenza virus genome. Sequence coverage for the different genome segments of wild type PR8 virus sequenced on Illumina MiSeq (2x250 bp, black lines, n = 2) or Ion Torrent PGM (Ion 318 chip v2, orange lines, n = 2). The obtained sequences were mapped to the reference genome (based on the pHW plasmids that were used to generate the virus, with addition of the extra 20 nucleotides present at the 5′ site in the RT-PCR primers).

    Techniques Used: Sequencing, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    30) Product Images from "Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing"

    Article Title: Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1284-z

    Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.
    Figure Legend Snippet: Next generation sequence analysis of pHW197-M. (A) Schematic representation of pHW197-M. HCMV: human cytomegalovirus promoter, T7: T7 RNA polymerase promoter, M1: matrix protein 1 open reading frame, M2: matrix protein 2 open reading frame (interrupted by an intron), hPolI: human RNA polymerase I promoter, pMB1 ori: origin of replication, Amp R : ampicillin resistance gene. (B) Mean sequencing depth after mapping the processed reads (n = 2) to the reference plasmid genome. The pHW197-M plasmid was fragmented with the Nextera XT DNA sample preparation kit before Illumina MiSeq sequence analysis or by Covaris mechanical shearing, followed by adaptor ligation before Ion Torrent PGM sequence analysis. (C) Percentage GC distribution in the pHW197-M plasmid reference sequence. The peak after position 2000 corresponds to the origin of replication.

    Techniques Used: Sequencing, Plasmid Preparation, Sample Prep, Ligation

    Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.
    Figure Legend Snippet: Quality of sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM platforms. The pHW197-M and pHW197-Mmut plasmids (= 7) were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or with Covaris mechanical shearing followed by adaptor ligation (Ion Torrent PGM). Distribution of the read lengths obtained on the Illumina MiSeq (A) and Ion Torrent PGM (B) before processing (in black, output files of sequencer) and after processing (in orange) the obtained sequencing reads. Processing implies removal of adaptor contamination, quality trimming ( > Q20), the removal of ambiguous bases and removal of reads shorter than 50 bases. For the Illumina MiSeq reads, broken pairs after read processing were also removed during the processing. Error bars represent the standard deviation. (C, D) Per-base quality distribution of sequencing reads. The Phred score distribution (Y-axis) relative to the processed reads obtained after sequencing on the Illumina MiSeq (C) and Ion Torrent PGM (D) . x% ile = x th percentile of quality scores observed at that position.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.
    Figure Legend Snippet: Comparison of nucleotide variants revealed by Illumina MiSeq and Ion torrent PGM sequencing. The pHW197-M and pHW197-Mmut plasmids were fragmented with the Nextera XT DNA sample preparation kit (Illumina MiSeq) or by Covaris mechanical shearing, followed by adaptor ligation (Ion Torrent PGM). The samples were sequenced in duplicate and the sequence reads were processed (adaptor removal, Q20 trimming, removal of ambiguous bases and removal of reads shorter than 50 bases). For reads obtained on the Illumina MiSeq: broken pairs after read processing were also removed. The relative percentages of substitutions, insertions and deletions were determined after mapping the processed Illumina MiSeq (A) and Ion Torrent PGM (B) sequencing reads to the pHW197-M (n = 2) or pHW197-Mmut (n = 2) reference sequence. Bars represent averages from 4 samples and error bars represent the standard deviation.

    Techniques Used: Sequencing, Sample Prep, Ligation, Standard Deviation

    31) Product Images from "Practical Synthesis of Cap‐4 RNA"

    Article Title: Practical Synthesis of Cap‐4 RNA

    Journal: Chembiochem

    doi: 10.1002/cbic.201900590

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    Figure Legend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Techniques Used: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    32) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    Figure Legend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
    Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
    Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    33) Product Images from "Profiling non-lysyl tRNAs in HIV-1"

    Article Title: Profiling non-lysyl tRNAs in HIV-1

    Journal: RNA

    doi: 10.1261/rna.1928110

    Detection of tRNAs by microarray analysis. ( A ) Array scheme. Total cellular or viral RNA were deacylated, and directly labeled with a Cy3 or Cy5 containing oligonucleotide using T4 DNA ligase. The labeling samples with the opposite fluorophores were combined
    Figure Legend Snippet: Detection of tRNAs by microarray analysis. ( A ) Array scheme. Total cellular or viral RNA were deacylated, and directly labeled with a Cy3 or Cy5 containing oligonucleotide using T4 DNA ligase. The labeling samples with the opposite fluorophores were combined

    Techniques Used: Microarray, Labeling

    34) Product Images from "Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends"

    Article Title: Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh761

    DNA-PKcs kinase activity regulates T4 DNA ligase-mediated DNA end joining. Purified DNA-PKcs (1 μg, lanes 10–15) and/or Ku (0.5 μg, lanes 6–8 and 13–15) were incubated in the absence (lanes 5 and 9) or presence (lanes 6–8 and 10–15) of T4-DNA ligase. Lanes 2–4 contained T4 DNA ligase alone (no Ku or DNA-PKcs). Lane 1 contained linearized ds plasmid DNA alone. Samples in lanes 4, 8, 12 and 15 were pre-incubated with wortmannin (W) (1 μM) or an equivalent volume of DMSO (D) (lanes 3, 7, 11 and 14). Reactions were stopped after 30 min and analyzed as described in Materials and Methods.
    Figure Legend Snippet: DNA-PKcs kinase activity regulates T4 DNA ligase-mediated DNA end joining. Purified DNA-PKcs (1 μg, lanes 10–15) and/or Ku (0.5 μg, lanes 6–8 and 13–15) were incubated in the absence (lanes 5 and 9) or presence (lanes 6–8 and 10–15) of T4-DNA ligase. Lanes 2–4 contained T4 DNA ligase alone (no Ku or DNA-PKcs). Lane 1 contained linearized ds plasmid DNA alone. Samples in lanes 4, 8, 12 and 15 were pre-incubated with wortmannin (W) (1 μM) or an equivalent volume of DMSO (D) (lanes 3, 7, 11 and 14). Reactions were stopped after 30 min and analyzed as described in Materials and Methods.

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

    Model for the role of DNA-PKcs autophosphorylation in DNA end ligation. In the first step on NHEJ, Ku (indicated by the small, closed circles) binds to the ends of the DSB. DNA-PKcs (large gray ellipses) is recruited to form the active DNA-PK complex. Upon synapsis, DNA-PK undergoes autophosphorylation, which causes a conformational change that renders the DNA ends accessible to end joining by T4 DNA ligase or the XRCC4/DNA-ligase IV complex.
    Figure Legend Snippet: Model for the role of DNA-PKcs autophosphorylation in DNA end ligation. In the first step on NHEJ, Ku (indicated by the small, closed circles) binds to the ends of the DSB. DNA-PKcs (large gray ellipses) is recruited to form the active DNA-PK complex. Upon synapsis, DNA-PK undergoes autophosphorylation, which causes a conformational change that renders the DNA ends accessible to end joining by T4 DNA ligase or the XRCC4/DNA-ligase IV complex.

    Techniques Used: Ligation, Non-Homologous End Joining

    35) Product Images from "The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T"

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

    Journal: Aging and Disease

    doi: 10.14336/AD.2016.0910

    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    Figure Legend Snippet: In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Techniques Used: In Vitro, Purification, Activity Assay, Staining, Western Blot, Positive Control

    36) Product Images from "In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts"

    Article Title: In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts

    Journal: Nucleic Acids Research

    doi:

    Differential repair of bleomycin-induced DSB ends in comparison with Stu I-induced blunt ends. Standard repair reactions were performed with either 100 ng substrate DNA linearized by 0.5 µg bleomycin/ml or Stu I digestion. The repair reactions contained either 15 µg PTN A, 2 U E.coli endonuclease IV (Endo IV), 5 U T4 DNA ligase (T4 Ligase) or combinations as indicated. Reactions were incubated at 17°C for 18 h. ( A ) The reactions were as follows: lane 1, bleomycin (blm)-damaged DNA negative control; lane 2, blm-damaged DNA + PTN A; lane 3, blm-damaged DNA + endo IV; lane 4, blm-damaged DNA + T4 ligase; lane 5, blm-damaged DNA + endo IV + T4 ligase; lane 6, Stu I cut (blunt-end) DNA negative control; lane 7, Stu I cut DNA + PTN A; lane 8, Stu I cut DNA + endo IV; lane 9, Stu I cut DNA + T4 ligase; lane 10, Stu I cut DNA + endo IV + T4 ligase. DNA substrate and product forms are indicated as linear (L), circular (C), dimer (D), trimer (T) and high molecular weight multimers (HM) to the right of the gel. ( B ) The data in the gel were plotted as a percentage of linear substrate DNA converted to rejoined products. Reactions performed with bleomycin-linearized DNA substrates are indicated by gray bars. Reactions conducted with the Stu I-linearized blunt-end DSB substrates are indicated by black bars.
    Figure Legend Snippet: Differential repair of bleomycin-induced DSB ends in comparison with Stu I-induced blunt ends. Standard repair reactions were performed with either 100 ng substrate DNA linearized by 0.5 µg bleomycin/ml or Stu I digestion. The repair reactions contained either 15 µg PTN A, 2 U E.coli endonuclease IV (Endo IV), 5 U T4 DNA ligase (T4 Ligase) or combinations as indicated. Reactions were incubated at 17°C for 18 h. ( A ) The reactions were as follows: lane 1, bleomycin (blm)-damaged DNA negative control; lane 2, blm-damaged DNA + PTN A; lane 3, blm-damaged DNA + endo IV; lane 4, blm-damaged DNA + T4 ligase; lane 5, blm-damaged DNA + endo IV + T4 ligase; lane 6, Stu I cut (blunt-end) DNA negative control; lane 7, Stu I cut DNA + PTN A; lane 8, Stu I cut DNA + endo IV; lane 9, Stu I cut DNA + T4 ligase; lane 10, Stu I cut DNA + endo IV + T4 ligase. DNA substrate and product forms are indicated as linear (L), circular (C), dimer (D), trimer (T) and high molecular weight multimers (HM) to the right of the gel. ( B ) The data in the gel were plotted as a percentage of linear substrate DNA converted to rejoined products. Reactions performed with bleomycin-linearized DNA substrates are indicated by gray bars. Reactions conducted with the Stu I-linearized blunt-end DSB substrates are indicated by black bars.

    Techniques Used: Incubation, Negative Control, Molecular Weight

    37) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.
    Figure Legend Snippet: Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.

    Techniques Used: Plasmid Preparation, Construct

    38) Product Images from "Engineering Adipose-like Tissue in vitro and in vivo Utilizing Human Bone Marrow and Adipose-derived Mesenchymal Stem Cells with Silk Fibroin 3D Scaffolds"

    Article Title: Engineering Adipose-like Tissue in vitro and in vivo Utilizing Human Bone Marrow and Adipose-derived Mesenchymal Stem Cells with Silk Fibroin 3D Scaffolds

    Journal:

    doi: 10.1016/j.biomaterials.2007.08.017

    Comparison of (FABP4), LPL, PPARgamma, GLUT4, adipsin, and ACS mRNA transcript levels of hASCs) seeded on aqueous based silk scaffolds (AB), HFIP-based silk scaffolds, collagen scaffolds (COL) and poly-lactic acid (PLA) by real-time RT-PCR analysis. hASCs
    Figure Legend Snippet: Comparison of (FABP4), LPL, PPARgamma, GLUT4, adipsin, and ACS mRNA transcript levels of hASCs) seeded on aqueous based silk scaffolds (AB), HFIP-based silk scaffolds, collagen scaffolds (COL) and poly-lactic acid (PLA) by real-time RT-PCR analysis. hASCs

    Techniques Used: Proximity Ligation Assay, Quantitative RT-PCR

    Comparison of FABP4, LPL, PPARgamma, GLUT4, adipsin, ACS mRNA transcript levels of hMSCs seeded on aqueous based silk scaffolds (AB), HFIP-based silk scaffolds (HF), collagen scaffolds (COL) and poly-lactic acid (PLA) by real-time RT-PCR analysis. hMSCs
    Figure Legend Snippet: Comparison of FABP4, LPL, PPARgamma, GLUT4, adipsin, ACS mRNA transcript levels of hMSCs seeded on aqueous based silk scaffolds (AB), HFIP-based silk scaffolds (HF), collagen scaffolds (COL) and poly-lactic acid (PLA) by real-time RT-PCR analysis. hMSCs

    Techniques Used: Proximity Ligation Assay, Quantitative RT-PCR

    39) Product Images from "Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)"

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    Journal: RNA

    doi: 10.1261/rna.5247704

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).
    Figure Legend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Techniques Used: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).
    Figure Legend Snippet: 5′-Adenylated RNA. ( A ) The structure of 5′-AppRNA. X is the 5′-terminal nucleotide of the RNA substrate before adenylation. ( B ) The T4 RNA ligase mechanism, showing the 5′-AppRNA intermediate 2 . X and X′ may be any nucleotides. ( C ) Nucleophilic displacement reaction on 5′-triphosphorylated RNA (5′-pppRNA). Nu, nucleophile. The 5′-terminal nucleotide of the RNA is shown as guanosine G because 5′-triphosphorylated RNAs are most typically prepared by in vitro transcription, which introduces G at this position. The nucleophilic substitution reaction on 5′-AppRNA is analogous, except with displacement of AMP instead of PP i (cf. 2 → 3 in B ).

    Techniques Used: In Vitro

    Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.
    Figure Legend Snippet: Possible reaction products from 5′-adenylation of an RNA substrate with T4 RNA ligase and ATP. 5′-monophosphate and 5′-adenyl pyrophosphate termini are abbreviated p and App, respectively. The 5′-to-3′ polarity of each strand is shown by an arrowhead pointing in the 3′-direction. The desired 5′-AppRNA is boxed, and the three possible side reactions starting from 5′-AppRNA are illustrated (circularization, oligomerization, and blocking oligo ligation). The abbreviations used for the other products in the remaining figures of this article are given in boldface within parentheses. For the oligomerization reaction, the RNA substrate that does not provide the reactive 5′-App may itself have either 5′-p or 5′-App. Therefore, two different oligomerization products of any given nucleotide length are possible; only one is shown here.

    Techniques Used: Blocking Assay, Ligation

    40) Product Images from "Studying RNA–DNA interactome by Red-C identifies noncoding RNAs associated with repressed chromatin compartment and reveals transcription dynamics"

    Article Title: Studying RNA–DNA interactome by Red-C identifies noncoding RNAs associated with repressed chromatin compartment and reveals transcription dynamics

    Journal: bioRxiv

    doi: 10.1101/859504

    The Red-C technique. ( A ) Outline of Red-C protocol. ( B ) Genomic distribution of DNA and RNA reads extracted from forward and reverse sequencing reads, respectively. As genic, we used RefSeq protein-coding genes that occupy 37% of the genome. Reads having the same direction as the transcript are defined as sense; reads having the opposite direction to the transcript are defined as antisense. ( C ) Correlation of RNA–DNA contacts with RNA-seq signal in K562 cells. Red line, linear regression. ( D ) RNA–DNA (Red-C) and DNA–DNA (K562 Hi-C ( Rao et al. 2014 )) contact matrices for a region of Chr 1 at a 100 Kb resolution. RNA-seq profile for K562 (1 Kb bins) and gene distribution are shown alongside. ( E ) Background profile in K562 cells. RPK, reads per Kb. ( F–J ) Fold enrichment of selected RNAs compared to the background in K562 cells ( F–I ) and female fibroblasts ( J ). MALAT profile is at 1 Kb resolution; the other profiles are at 100 Kb resolution.
    Figure Legend Snippet: The Red-C technique. ( A ) Outline of Red-C protocol. ( B ) Genomic distribution of DNA and RNA reads extracted from forward and reverse sequencing reads, respectively. As genic, we used RefSeq protein-coding genes that occupy 37% of the genome. Reads having the same direction as the transcript are defined as sense; reads having the opposite direction to the transcript are defined as antisense. ( C ) Correlation of RNA–DNA contacts with RNA-seq signal in K562 cells. Red line, linear regression. ( D ) RNA–DNA (Red-C) and DNA–DNA (K562 Hi-C ( Rao et al. 2014 )) contact matrices for a region of Chr 1 at a 100 Kb resolution. RNA-seq profile for K562 (1 Kb bins) and gene distribution are shown alongside. ( E ) Background profile in K562 cells. RPK, reads per Kb. ( F–J ) Fold enrichment of selected RNAs compared to the background in K562 cells ( F–I ) and female fibroblasts ( J ). MALAT profile is at 1 Kb resolution; the other profiles are at 100 Kb resolution.

    Techniques Used: Sequencing, RNA Sequencing Assay, Hi-C

    MIR3648 and MIR3687 target inactive chromatin. ( A ) Coverage of the MIR3648/3867 locus by RNA parts of RNA–DNA chimeras. RNA parts are displayed in the stack view in accordance with mapping coordinates of RNA 5’ and 3’ ends. ( B,C ) Frequency of contacts of MIR3648, MIR3687, and U2 with different chromatin types ( B ) and A and B spatial compartments ( C ) determined for the full genome. The maximal contact frequency for a given RNA is taken to be equal to 1. Error bars, SEM for two biological replicates. Active chromatin is defined as combination of types 1, 2, 4, 5, 6, 7, 9, 10, and 11; Polycomb, of types 3 and 12; Heterochromatin, of type 13. A/B compartment track for K562 was obtained from ( Rao et al. 2014 ). ( D ) Frequency of contacts of MIR3648, MIR3687, and U2 with expressed protein-coding genes (divided into three equal groups based on the density of RNA-seq signal), non-expressed protein-coding genes (RNA-seq signal = 0), and gene deserts (regions of > 500 Kb not occupied by any genes). For each RNA, the total number of contacts with genes of each group and gene deserts was determined, normalized by the total length of genes in the group and gene deserts, and presented relative to the maximal value for a given RNA (taken equal to 1). ( E ) Contacts of MIR3648, MIR3687, and U2 with 1 Mb genomic bins divided into five equal groups based on RNA-seq signal in the bin ( n = 576, p -values are from Tukey’s multiple comparisons test). Bins occupied by chromatin types 1–13 by less than 10% are not included in the analysis. RPK, reads per Kb; RPM, reads per Mb. ( F ) Distribution of raw contacts of MIR3687 along Chrs 18 and 19 and fold enrichment compared to background at a 50 Kb resolution. Gene distribution, RNA-seq signal (1 Kb bin), and replication timing profile for K562 as determined by Repli-seq ( Hansen et al. 2010 ) are shown. ( G ) Distribution of correlation coefficients upon comparison of MIR3687 fold enrichment profile with Repli-seq in genomic windows of 20 Mb, as examined by StereoGene ( Stavrovskaya et al. 2017 ). The genome-wide correlation coefficients calculated with the kernel and p -values are presented. ( H ) Fold enrichment of MIR3648, MIR3687, and U2 at individual chromosomes relative to overall contact frequency of respective RNAs in the genome. Error bars, SEM for two biological replicates.
    Figure Legend Snippet: MIR3648 and MIR3687 target inactive chromatin. ( A ) Coverage of the MIR3648/3867 locus by RNA parts of RNA–DNA chimeras. RNA parts are displayed in the stack view in accordance with mapping coordinates of RNA 5’ and 3’ ends. ( B,C ) Frequency of contacts of MIR3648, MIR3687, and U2 with different chromatin types ( B ) and A and B spatial compartments ( C ) determined for the full genome. The maximal contact frequency for a given RNA is taken to be equal to 1. Error bars, SEM for two biological replicates. Active chromatin is defined as combination of types 1, 2, 4, 5, 6, 7, 9, 10, and 11; Polycomb, of types 3 and 12; Heterochromatin, of type 13. A/B compartment track for K562 was obtained from ( Rao et al. 2014 ). ( D ) Frequency of contacts of MIR3648, MIR3687, and U2 with expressed protein-coding genes (divided into three equal groups based on the density of RNA-seq signal), non-expressed protein-coding genes (RNA-seq signal = 0), and gene deserts (regions of > 500 Kb not occupied by any genes). For each RNA, the total number of contacts with genes of each group and gene deserts was determined, normalized by the total length of genes in the group and gene deserts, and presented relative to the maximal value for a given RNA (taken equal to 1). ( E ) Contacts of MIR3648, MIR3687, and U2 with 1 Mb genomic bins divided into five equal groups based on RNA-seq signal in the bin ( n = 576, p -values are from Tukey’s multiple comparisons test). Bins occupied by chromatin types 1–13 by less than 10% are not included in the analysis. RPK, reads per Kb; RPM, reads per Mb. ( F ) Distribution of raw contacts of MIR3687 along Chrs 18 and 19 and fold enrichment compared to background at a 50 Kb resolution. Gene distribution, RNA-seq signal (1 Kb bin), and replication timing profile for K562 as determined by Repli-seq ( Hansen et al. 2010 ) are shown. ( G ) Distribution of correlation coefficients upon comparison of MIR3687 fold enrichment profile with Repli-seq in genomic windows of 20 Mb, as examined by StereoGene ( Stavrovskaya et al. 2017 ). The genome-wide correlation coefficients calculated with the kernel and p -values are presented. ( H ) Fold enrichment of MIR3648, MIR3687, and U2 at individual chromosomes relative to overall contact frequency of respective RNAs in the genome. Error bars, SEM for two biological replicates.

    Techniques Used: RNA Sequencing Assay, Genome Wide

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