site-specific recombination Thermo Fisher Search Results


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  • 99
    Thermo Fisher dynabeads protein g
    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with <t>Protein-G</t> <t>Dynabeads</t> (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei
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    Thermo Fisher topo ta cloning kit
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
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    Thermo Fisher cdna ends
    Polysome analysis of <t>Gata4</t> E1a and E1b transcripts in the testis. RNA from adult mouse testis was centrifuged through a linear sucrose gradient to separate transcripts associated with monoribosomes (not actively translated) and polyribosomes (actively translated). Monoribosomal and polyribosomal fractions are found at the top and towards the bottom of the gradient, respectively. Twelve fractions were collected from the gradient, the RNA was extracted from each fraction, reverse-transcribed into <t>cDNA</t> and subjected to PCR with primers specific for Gata4 E1a and E1b. Both transcripts are associated with the polyribosomal fractions indicating that they are being actively translated into protein. EDTA, which sequesters magnesium ions required for ribosome stability, was used as a control. In the presence of EDTA, the transcript profiles show a characteristic shift to the top of the gradient indicating a successful dissociation of the polyribosomes.
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    Thermo Fisher zero blunt topo pcr cloning kit
    Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in <t>PCR-TOPO</t> sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region
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    Thermo Fisher dynabeads protein a
    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using <t>Dynabeads</t> <t>Protein</t> A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004
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    Thermo Fisher lr clonase ii
    The rationale of BioVector. The LR reaction among GEC, PEC and BDV shows the basic principle of BioVector, in which a gene and a promoter in individual entry clones are positioningly and directionally joined together in a destination vector. att L1/2/3/4, att R1/2/3/4, and att P1/2/3/4, recombination sites; ccd B, a negative selection marker for DH5α; LR <t>Clonase</t> II®, recombination enzyme from Invitrogen.
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    Thermo Fisher dynabeads m 280 streptavidin
    Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to <t>Dynabeads/M-280</t> <t>streptavidin</t> and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.
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    Thermo Fisher gateway lr clonase ii enzyme mix
    Map of pGADT7-DEST. This plasmid was modified from pGADT7-Rec (Clontech Laboratories); the SMART III and CDS III sequences were replaced by attR1 and attR2, respectively. Following recombination catalysis using the Gateway ® LR <t>Clonase</t> ™ II enzyme mix (Invitrogen), attR1-Cm-ccdB-attR2 was replaced by attB1-cDNA insert-attB2.
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    Thermo Fisher actev protease
    Tandem affinity purification (TAP) and proteomic analysis of TbMCU complex. (A) Diagram showing positions of tags. <t>TEV,</t> tobacco etch virus protease cleavage site; MH, Myc-His; CBP, calmodulin-binding peptide; Prot A, protein A domain. (B) Scheme of the TAP method used for isolation of TAP-tagged TbMCU complex. (C) Western blot analyses of tetracycline-inducible TAP-tagged TbMCU-TAP overexpressed in PCF trypanosomes against the CBP, MYC, and TbMCU antibodies. One additional band at approximately 40 kDa was detected, possibly because of degradation of the TAP-tagged TbMCU protein (arrows) by the <t>AcTEV</t> protease or an alternative translation termination at the TEV cleavage site. When anti-TbMCU antibody was used, both the TAP-tagged protein and the endogenous copy were detected. The blot was stripped and reincubated with antitubulin (α-Tub) antibody as a loading control. (D) SDS-PAGE of purified TbMCU complex (left, silver stained [SS]; right, immunoblotted with anti-TbMCU). M, PageRuler unstained protein ladder.
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    Thermo Fisher protein g sepharose
    vRNA binding by RIG-I. GFP-RIG-I expressed by 293T cells was coupled to <t>protein</t> G <t>Sepharose</t> beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H 2 O was used as negative control.
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    Thermo Fisher streptavidin beads
    Analysis of the capability of Sen1 variants to terminate transcription in vitro . ( A ) Scheme of an in vitro transcription termination assay. A schematic of a ternary EC based on previous biochemical and structural analyses ( 47 , 48 ) is shown on the top. Promoter-independent assembly of ECs is performed using a 9 nt RNA:DNA hybrid that occupies the RNAPII catalytic center. Ternary ECs are attached to <t>streptavidin</t> beads via the 5΄ biotin of the non-template strand allowing subsequent separation of bead-associated (B) and supernatant (S) fractions. The RNA is fluorescently labeled with FAM at the 5΄-end. The transcription template contains a G-less cassette followed by a G-stretch in the non-template strand. After adding an ATP, UTP, CTP mix, the RNAPII transcribes until it encounters the G-rich sequence. Sen1 provokes dissociation of ECs paused at the G-rich stretch and therefore the release of RNAPII and associated transcripts to the supernatant. ( B ) Denaturing PAGE analysis of RNAs from a representative IVTer assay in the absence and in the presence of Sen1 proteins. ( C ) Quantification of the fraction of transcripts released from ECs stalled at the G-stretch as a measure of the termination efficiency. Values represent the average and standard deviation of three independent experiments. The p-value associated with a t -test (p) is indicated.
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    Thermo Fisher genetailor site directed mutagenesis system
    Analysis of the capability of Sen1 variants to terminate transcription in vitro . ( A ) Scheme of an in vitro transcription termination assay. A schematic of a ternary EC based on previous biochemical and structural analyses ( 47 , 48 ) is shown on the top. Promoter-independent assembly of ECs is performed using a 9 nt RNA:DNA hybrid that occupies the RNAPII catalytic center. Ternary ECs are attached to <t>streptavidin</t> beads via the 5΄ biotin of the non-template strand allowing subsequent separation of bead-associated (B) and supernatant (S) fractions. The RNA is fluorescently labeled with FAM at the 5΄-end. The transcription template contains a G-less cassette followed by a G-stretch in the non-template strand. After adding an ATP, UTP, CTP mix, the RNAPII transcribes until it encounters the G-rich sequence. Sen1 provokes dissociation of ECs paused at the G-rich stretch and therefore the release of RNAPII and associated transcripts to the supernatant. ( B ) Denaturing PAGE analysis of RNAs from a representative IVTer assay in the absence and in the presence of Sen1 proteins. ( C ) Quantification of the fraction of transcripts released from ECs stalled at the G-stretch as a measure of the termination efficiency. Values represent the average and standard deviation of three independent experiments. The p-value associated with a t -test (p) is indicated.
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    Thermo Fisher protein g sepharose beads
    Features of Drosophila U7 snRNA. ( A ) The sequence and a possible secondary structure of 71-nt Drosophila U7 snRNA. The putative Sm-binding site is underlined. ( B ) Comparison of the Sm-binding site from four previously identified U7 snRNAs and from Drosophila U7 snRNA. The consensus sequence for the previously identified U7 snRNAs is shown and the invariant nucleotides in the U7 snRNA of all four species are boxed. The arrowhead indicates the nucleotide in Drosophila Sm-binding site that departs from the consensus. ( C ) RNA extracted from a Kc nuclear extract (lane 1) was incubated with either a nonspecific mAb (lane 2) or αTMG Ab (lane 3), and the RNA was recovered from protein <t>G-Sepharose</t> and analyzed by Northern blotting. A small amount of 32 P-labeled 86-nt RNA (asterisk) was added to each sample before ethanol precipitation to control for the efficiency of RNA recovery.
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    Thermo Fisher dynabeads protein g immunoprecipitation kit
    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein <t>G-dynabeads</t> and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.
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    Thermo Fisher dynabeads myone streptavidin c1
    Biotin affinity purification and western blot analysis. <t>Dynabeads</t> <t>MyOne</t> <t>Streptavidin</t> C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,
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    Thermo Fisher ta cloning kit
    Biotin affinity purification and western blot analysis. <t>Dynabeads</t> <t>MyOne</t> <t>Streptavidin</t> C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,
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    Thermo Fisher lipofectamine 2000
    Effect of Foxa1 overexpression or deficiency on A549 cell apoptosis induced by H 2 O 2 . a A549 cells were transfected with pcDNA3.1-Foxa1 and the expression levels of Foxa1 were assessed by Western blotting. Ctrl A549 cells treated with <t>lipofectamine</t> only,
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    Thermo Fisher block it pol ii mir rnai expression vector kit
    Silencing of PLB in CM by <t>amiR155-PLBr</t> and shPLBr at the mRNA and protein level. (A) Dose dependence of <t>RNAi-mediated</t> PLB mRNA silencing detected by Northern blot analysis. CM were transduced with either scAAV6-amiR155-PLBr (amiR155-PLBr), scAAV6-shPLBr (shPLBr) or control AAV vector (amiR155-Con) and the cells were harvested 7 days later. The multiple bands represent alternatively spliced PLB mRNA transcripts of different lengths. (B) Dose dependence of RNAi-mediated PLB silencing at the protein level for transduction conditions described under A. Upper panel : Representative Western blots showing the immunoreactive signals of PLB and GAPDH. Lower panel : Results of the semi-quantitative analysis of the blots. The obtained PLB/GAPDH signal ratio values were normalized to that of non-transduced cells (no add). (C-E) Time dependence of RNAi-mediated PLB silencing. CM were transduced with 25×10 3 vg/c of scAAV6-shPLBr, scAAV6-amiR155-PLBr, scAAV6-shCon, or scAAV6-amiR155-Con as indicated and then analyzed at indicated time points. (C) The PLB mRNA expression levels were determined by qRT-PCR. Quantification of relative PLB mRNA levels was done as described under ( Figure 2B ). (D) Representative Western blot of PLB, SERCA2a and GAPDH. (E) Results of the semi-quantitative Western blotting analysis of four independent experiments. The bars indicate the percentage of PLB ( upper panel ) and SERCA2a ( lower panel ) abundance found in scAAV6-shPLBr (shPLBr) and scAAV6-amiR155-PLBr (amiR155-PLBr) vector transduced cells as compared to respective control vector transduced CM exhibiting PLB and SERCA2a abundance of 100%, respectively. *p
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    Thermo Fisher single stranded dna
    EV proteins and <t>RNA</t> are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded <t>DNA</t> (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
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    Image Search Results


    Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Expression of reporters and capture SUN1mRFP1Flag tagged nuclei from mature 3T3-L1 adipocytes. a Six days after ADNp::mRFP1 nucleofected 3T3-L1 preadipocytes were induced to differentiate into MAs strong cytoplasmic fluorescence of the mRFP1 reporter was detected (see Table 1 , Additional file 2 : Figure S2). Pre-adipocytes did not have detectable fluorescence. This result shows the promoter vector was specifically expressed in MAs. b mRFP1 fluorescence from the ADNp::SUN1mRFP1Flag reporter on the surface of differentiated 3T3-L1 nuclei selected from c. c Expression of the ADNp::SUN1mRFP1Flag reporter in MAs 10 days after induction of nucleofected 3T3-L1 preadipocytes (DAPI stained DNA ( blue ), lipid rich oil droplets detected with Lipotox Green ( green ), enhanced red fluorescent protein mRFP1 ( red ). The lower right hand panel shows the merged fluorescence image. mRFP1 fluorescence is associated with the nuclei. It should be noted that spherical oil droplets often produce an optical distortion of adjacent nuclei. d Adipocyte nuclei from mature 3T3-L1 cells expressing the SUN1mRFP1Flag reporter were captured with Protein-G Dynabeads (2.8 μm diameter) pre-adsorbed with anti-GFP antibody. e A negative control capture performed in parallel using Protein-G Dynabeads pre-adsorbed with anti-CD4 T cell specific antibody did not capture nuclei

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Expressing, Fluorescence, Plasmid Preparation, Staining, Negative Control

    Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Journal: BMC obesity

    Article Title: Adipocyte nuclei captured from VAT and SAT

    doi: 10.1186/s40608-016-0112-6

    Figure Lengend Snippet: Mature MA-INTACT mouse adipocyte nuclei expressed SUN1mRFP1Flag RNA in BAT, VAT, and SAT allowing adipocyte nuclei to be captured on Immuno-paramagnetic beads. a Illustration of mouse fat deposits examined. b , c , d , e . Nuclei were efficiently captured from crude nuclear preparations prepared from inguinal SAT ( b ) retroperitioneal VAT ( c ) epididymal VAT ( d ) and scapular BAT ( e ). Protein G Dynabeads pre-adsorbed to rabbit anti-mRFP1 polyclonal antibody were used in these capture experiments. The clumping of nuclei occurs during successive rounds of washing and capture. Negative control antibodies did not capture significant numbers of nuclei (not shown). f A Western blot ( top panel ) showed that preparations of captured VAT, SAT, and BAT nuclei expressed the 131 kDa SUN1mRFP1Flag reporter protein, while uncaptured nuclei did not. In house prepared anti-mRFP1 rabbit pAb detected the mRFP1 domain. Purified 25 kDa mRFP1 was run as a positive control. A loading control ( bottom panel ) showed the approximately equal loading of nuclear proteins and an mRFP1 standard. The loading control samples were run for a short time (20 min) on an SDS PAGE system and protein front stained with Coomassie

    Article Snippet: Immuno-capture of nuclei Anti-RFP, anti-FLAG, anti-CD4, anti-CD8, anti-mRFP1 antibodies were coupled to 2.8 micron diameter protein G supraparamagnetic Dynabeads (ThermoFischer Scientific, #10004D).

    Techniques: Negative Control, Western Blot, Purification, Positive Control, SDS Page, Staining

    ITGA5 trafficking in ECs is NRP2 dependent. ( A ): Label-free quantitative mass spectrometry peptide hits identified using MaxQuant software from the Andromeda peptide database. Panel depicts peptide hits detected at a higher fold-change in two control siRNA treated EC lines than those detected from two NRP2 siRNA treated EC lines. NRP2 silencing was confirmed by subjecting EC extracts to Western blot analysis. ( B ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. EC extracts were immunoprecipitated by incubation with protein-G Dynabeads® coupled to a NRP2 antibody. Immunoprecipitated complexes were subjected to Western blot analysis using antibodies against clathrin heavy chain-1 (top) and caveolin-1 (bottom). ( C ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. ECs were subsequently starved in serum-free media, before being placed on ice. EC cell surface proteins were labelled with 0.3 mg/ml biotin. ECs were then incubated for the indicated timepoints at 37°C, 5% CO 2 . A sample of ECs were maintained at 4°C for use as positive/negative (+/-Mesna) controls. Following incubation at 37°C, ECs were placed on ice and incubated with 100 mM Mesna. EC lysates were then immunoprecipitated with protein-G Dynabeads® coupled to an anti-biotin antibody. Immunoprecipitated biotin-labelled proteins were separated by SDS-PAGE and subjected to Western bolt analysis. The level of internalised ITGA5 at each time of incubation was normalised to the (-) Mesna control. Left panel shows Western blot image, right panel shows the mean densitometric analysis obtained using ImageJ TM . Also shown is confirmation of NRP-2 silencing. N=3 independent experiments. ( D ): Co-immunoprecipitation study as described in B . Immunoprecipitated complexes were subjected to Western blot analysis using an anti-Rab11 antibody. ( E ): siRNA-transfected ECs were prepared as described in C . After biotin surface labelling, ECs were incubated in serum free media for 20 minutes at 37°C to allow for internalisation. A sample of ECs were maintained at 4°C for use as positive/negative controls. The remaining ECs were then placed on ice, and any un-internalised biotin-labelled proteins stripped off using 100 mM Mesna. The internalised protein fraction was allowed to recycle by incubating the ECs for the indicated timepoints at 37°C. ECs were then returned to ice and incubated in 100 mM Mesna. No Mesna treatment dishes at each timepoint were used as comparative controls. All subsequent stages were performed in the same manner as described in C . The level of the recycled ITGA5 was determined by normalising the amount of ITGA5 quantified from dishes treated with Mesna to the total ITGA5 on the membranes of the Mesna-untreated cells in the same period of incubation. N=3 independent experiments.

    Journal: bioRxiv

    Article Title: NRP2 as an emerging angiogenic player; promoting endothelial cell adhesion and migration by regulating recycling of α5 integrin

    doi: 10.1101/744763

    Figure Lengend Snippet: ITGA5 trafficking in ECs is NRP2 dependent. ( A ): Label-free quantitative mass spectrometry peptide hits identified using MaxQuant software from the Andromeda peptide database. Panel depicts peptide hits detected at a higher fold-change in two control siRNA treated EC lines than those detected from two NRP2 siRNA treated EC lines. NRP2 silencing was confirmed by subjecting EC extracts to Western blot analysis. ( B ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. EC extracts were immunoprecipitated by incubation with protein-G Dynabeads® coupled to a NRP2 antibody. Immunoprecipitated complexes were subjected to Western blot analysis using antibodies against clathrin heavy chain-1 (top) and caveolin-1 (bottom). ( C ): siRNA-transfected ECs were seeded onto FN and incubated for 48 hours. ECs were subsequently starved in serum-free media, before being placed on ice. EC cell surface proteins were labelled with 0.3 mg/ml biotin. ECs were then incubated for the indicated timepoints at 37°C, 5% CO 2 . A sample of ECs were maintained at 4°C for use as positive/negative (+/-Mesna) controls. Following incubation at 37°C, ECs were placed on ice and incubated with 100 mM Mesna. EC lysates were then immunoprecipitated with protein-G Dynabeads® coupled to an anti-biotin antibody. Immunoprecipitated biotin-labelled proteins were separated by SDS-PAGE and subjected to Western bolt analysis. The level of internalised ITGA5 at each time of incubation was normalised to the (-) Mesna control. Left panel shows Western blot image, right panel shows the mean densitometric analysis obtained using ImageJ TM . Also shown is confirmation of NRP-2 silencing. N=3 independent experiments. ( D ): Co-immunoprecipitation study as described in B . Immunoprecipitated complexes were subjected to Western blot analysis using an anti-Rab11 antibody. ( E ): siRNA-transfected ECs were prepared as described in C . After biotin surface labelling, ECs were incubated in serum free media for 20 minutes at 37°C to allow for internalisation. A sample of ECs were maintained at 4°C for use as positive/negative controls. The remaining ECs were then placed on ice, and any un-internalised biotin-labelled proteins stripped off using 100 mM Mesna. The internalised protein fraction was allowed to recycle by incubating the ECs for the indicated timepoints at 37°C. ECs were then returned to ice and incubated in 100 mM Mesna. No Mesna treatment dishes at each timepoint were used as comparative controls. All subsequent stages were performed in the same manner as described in C . The level of the recycled ITGA5 was determined by normalising the amount of ITGA5 quantified from dishes treated with Mesna to the total ITGA5 on the membranes of the Mesna-untreated cells in the same period of incubation. N=3 independent experiments.

    Article Snippet: 100 µg protein from each sample was immunoprecipitated by incubation with protein-G Dynabeads® (Invitrogen) coupled to a rabbit anti-NRP2 antibody (clone 3366, Cell Signalling Technology) on a rotator overnight at 4°C.

    Techniques: Mass Spectrometry, Software, Western Blot, Transfection, Incubation, Immunoprecipitation, SDS Page

    FMRP promotes AKAP Rugose expression in the MB learning/memory circuit. (A) RNA immunoprecipitation (RIP) comparing w 1118 genetic background control and dfmr1 null brains ( dfmr1 50M / dfmr1 50M ). Amplified cDNA transcribed from input lysates and RNA pulled down with anti-FMRP-conjugated Dynabeads; futsch is the positive control and α- tubulin is the negative control. Molecular weights are indicated to the right. (B) Western blots of control ( w 1118 ) vs. dfmr1 null ( dfmr1 50M )(top), and driver control ( elav- Gal4/+) vs. UAS- dfmr1 rescue ( elav- Gal4/+; UAS- dfmr1 9557−3 /+; dfmr1 50M /dfmr1 50M ) bottom) brains at 1 day post-eclosion (1dpe). Proteins indicated on left, molecular weights on the right. (C-D) Dot plots of the normalized Rugose band intensities, showing the mean ± SEM. (E) Representative confocal images of anti-Rugose in w 1118 control and dfmr1 null brains. Dashed lines separate MB Kenyon cell somata from surrounding brain tissue. (F) Dot plots of normalized Rugose intensity, showing mean ± SEM. (G) Representative confocal images of anti-Rugose in MB-specific driver control (OK107-Gal4/+) and FMRP overexpression (OE) in MB (UAS- dfmr1 9557−3 /+; OK107-G4/+). Dashed lines separate the MB Kenyon cell somata from surrounding brain tissue. (H) Dot plots of normalized Rugose intensity, showing mean ± SEM. Statistics were done with unpaired Welch’s t-tests. Statistical significance is indicated as n.s. (not significant) and *** (p

    Journal: Neurobiology of disease

    Article Title: Fragile X Mental Retardation Protein Positively Regulates PKA Anchor Rugose and PKA Activity to Control Actin Assembly in Learning/Memory Circuitry

    doi: 10.1016/j.nbd.2019.02.004

    Figure Lengend Snippet: FMRP promotes AKAP Rugose expression in the MB learning/memory circuit. (A) RNA immunoprecipitation (RIP) comparing w 1118 genetic background control and dfmr1 null brains ( dfmr1 50M / dfmr1 50M ). Amplified cDNA transcribed from input lysates and RNA pulled down with anti-FMRP-conjugated Dynabeads; futsch is the positive control and α- tubulin is the negative control. Molecular weights are indicated to the right. (B) Western blots of control ( w 1118 ) vs. dfmr1 null ( dfmr1 50M )(top), and driver control ( elav- Gal4/+) vs. UAS- dfmr1 rescue ( elav- Gal4/+; UAS- dfmr1 9557−3 /+; dfmr1 50M /dfmr1 50M ) bottom) brains at 1 day post-eclosion (1dpe). Proteins indicated on left, molecular weights on the right. (C-D) Dot plots of the normalized Rugose band intensities, showing the mean ± SEM. (E) Representative confocal images of anti-Rugose in w 1118 control and dfmr1 null brains. Dashed lines separate MB Kenyon cell somata from surrounding brain tissue. (F) Dot plots of normalized Rugose intensity, showing mean ± SEM. (G) Representative confocal images of anti-Rugose in MB-specific driver control (OK107-Gal4/+) and FMRP overexpression (OE) in MB (UAS- dfmr1 9557−3 /+; OK107-G4/+). Dashed lines separate the MB Kenyon cell somata from surrounding brain tissue. (H) Dot plots of normalized Rugose intensity, showing mean ± SEM. Statistics were done with unpaired Welch’s t-tests. Statistical significance is indicated as n.s. (not significant) and *** (p

    Article Snippet: Briefly: 100 staged 1dpe heads from w 1118 control and dfmr1 50M null animals were homogenized in lysis buffer (20 mM HEPES, 100 mM NaCl, 2.5 mM EDTA, 0.05% Triton X, 5% glycerol, 1X SigmaFast Protease inhibitor (Sigma S8820), 120 Units/mL Protector RNase inhibitor (Roche 03335399001), then incubated overnight at 4°C with Dynabeads (Invitrogen 10003D) pre-conjugated to mouse anti-FMRP (Abcam 6A15) following the manufacturer’s protocols.

    Techniques: Expressing, Immunoprecipitation, Amplification, Positive Control, Negative Control, Western Blot, Over Expression

    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Journal: Disease Models & Mechanisms

    Article Title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    doi: 10.1242/dmm.026765

    Figure Lengend Snippet: Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Article Snippet: The resulting PCRs from the same group of fish were checked on the gel, mixed, purified using QIAquick PCR purification kit (QIAGEN, 28104) and TOPO-cloned into pCR2.1-TOPO (Thermo Fisher Scientific, 450641).

    Techniques: Mutagenesis, Sequencing, Fluorescence In Situ Hybridization, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Polysome analysis of Gata4 E1a and E1b transcripts in the testis. RNA from adult mouse testis was centrifuged through a linear sucrose gradient to separate transcripts associated with monoribosomes (not actively translated) and polyribosomes (actively translated). Monoribosomal and polyribosomal fractions are found at the top and towards the bottom of the gradient, respectively. Twelve fractions were collected from the gradient, the RNA was extracted from each fraction, reverse-transcribed into cDNA and subjected to PCR with primers specific for Gata4 E1a and E1b. Both transcripts are associated with the polyribosomal fractions indicating that they are being actively translated into protein. EDTA, which sequesters magnesium ions required for ribosome stability, was used as a control. In the presence of EDTA, the transcript profiles show a characteristic shift to the top of the gradient indicating a successful dissociation of the polyribosomes.

    Journal: PLoS ONE

    Article Title: Conserved Usage of Alternative 5? Untranslated Exons of the GATA4 Gene

    doi: 10.1371/journal.pone.0008454

    Figure Lengend Snippet: Polysome analysis of Gata4 E1a and E1b transcripts in the testis. RNA from adult mouse testis was centrifuged through a linear sucrose gradient to separate transcripts associated with monoribosomes (not actively translated) and polyribosomes (actively translated). Monoribosomal and polyribosomal fractions are found at the top and towards the bottom of the gradient, respectively. Twelve fractions were collected from the gradient, the RNA was extracted from each fraction, reverse-transcribed into cDNA and subjected to PCR with primers specific for Gata4 E1a and E1b. Both transcripts are associated with the polyribosomal fractions indicating that they are being actively translated into protein. EDTA, which sequesters magnesium ions required for ribosome stability, was used as a control. In the presence of EDTA, the transcript profiles show a characteristic shift to the top of the gradient indicating a successful dissociation of the polyribosomes.

    Article Snippet: 5′ RACE Transcriptional start sites of the GATA4 gene were mapped by rapid amplification of cDNA ends (5′ RACE) using the GeneRacer kit according to instructions supplied by the manufacturer (Invitrogen).

    Techniques: Polymerase Chain Reaction

    Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region

    Journal: Nature Communications

    Article Title: Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses

    doi: 10.1038/s41467-017-02706-7

    Figure Lengend Snippet: Recombinant AAV vectors can mediate homology-directed repair (HDR). a Schematic representation of the Tyr locus and location of sgRNA in exon 1. The orange and red lines mark the initiation and termination codons respectively. The green line indicates the location of the sgRNA used to target Tyr . b Strategy to introduce a premature stop codon in the Tyr locus using HDR. The 5′ and 3′ homology arms are marked by a thick line. A G to T nucleotide transversion in the PAM sequence converts a glycine codon (GGA) into a stop codon (TGA) disrupting translation of Tyr . Arrows indicate binding sites of the primers used in PCR-TOPO sequencing. c Strategy to insert the blue fluorescent protein (BFP) gene into the Tyr locus using HDR. Brown and purple arrows depict the binding sites of PCR primers used to confirm the insertion of BFP into Tyr locus. P2A, Porcine teschovirus-1 2A peptide; TAA, Stop codon. d Histogram showing the frequency of single-nucleotide transversion and BFP insertion by HDR using two different mixtures of rAAV vectors. e Analysis of single-nucleotide transversion in individual embryos or pups using PCR-TOPO sequencing. Each bar represents an individual sample. For pups, only DNA from tail snips and ear punches was analyzed. f Confirmation of BFP insertion using PCR. Four out of seven E3.5 embryos tested showed correct insertion of BFP into the Tyr locus. The top panel shows amplification of the 5′-junction of the targeted Tyr locus using a forward primer that binds to genomic DNA upstream of the homology region and a reverse primer that binds to the BFP gene as shown in ( c ). The bottom panel shows amplification of the 3′-junction of the Tyr- edited allele using a forward primer that binds to the BFP gene and a reverse primer that binds to genomic DNA downstream of the homology region

    Article Snippet: Purified PCR products were cloned into the pCRTM -Blunt II-TOPO vector using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Sci.

    Techniques: Recombinant, Introduce, Sequencing, Binding Assay, Polymerase Chain Reaction, Amplification

    EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Journal: eLife

    Article Title: Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis

    doi: 10.7554/eLife.00675

    Figure Lengend Snippet: EIN3 antibody reproducibly enriches DNA in chromatin immunoprecipitation. ( A ) Enrichment of the known target of EIN3, the promoter of ERF1, using Dynabeads Protein A and Dynabeads Sheep anti-Rabbit IgG to collect protein-DNA complexes. The average fold change for two technical ChIP replicates with three QPCR technical replicates each is shown. ( B ) Reproducibility in the two biological replicates for EIN3 ChIP performed upon treatment of ethylene gas for 0, 0.5, 1, and 4 hr. ( C ) Average RPKM of EIN3 binding sites 0, 0.5, 1, and 4 hr of ethylene gas treatment. ( D ) EIN3 binding preferentially occurs in the promoter regions of genes (1000 bp upstream of the TSS). DOI: http://dx.doi.org/10.7554/eLife.00675.004

    Article Snippet: We then substituted Dynabeads Protein A (Invitrogen, cat#100-1D) and Dynabeads M-280 Sheep anti-Rabbit IgG (Invitrogen, cat#112-04D) for the salmon sperm DNA blocked Protein A agarose beads recommended in the protocol (4), as to avoid sequencing of salmon sperm DNA.

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

    The rationale of BioVector. The LR reaction among GEC, PEC and BDV shows the basic principle of BioVector, in which a gene and a promoter in individual entry clones are positioningly and directionally joined together in a destination vector. att L1/2/3/4, att R1/2/3/4, and att P1/2/3/4, recombination sites; ccd B, a negative selection marker for DH5α; LR Clonase II®, recombination enzyme from Invitrogen.

    Journal: BMC Plant Biology

    Article Title: BioVector, a flexible system for gene specific-expression in plants

    doi: 10.1186/1471-2229-13-198

    Figure Lengend Snippet: The rationale of BioVector. The LR reaction among GEC, PEC and BDV shows the basic principle of BioVector, in which a gene and a promoter in individual entry clones are positioningly and directionally joined together in a destination vector. att L1/2/3/4, att R1/2/3/4, and att P1/2/3/4, recombination sites; ccd B, a negative selection marker for DH5α; LR Clonase II®, recombination enzyme from Invitrogen.

    Article Snippet: 5 μL reaction system was used for multiple-components LR, including Fu39-2 (20 ~ 50 ng), two entry clones (120 ~ 150ng, respectively) and 1 μL LR Clonase II Enzyme mix (Invitrogen).

    Techniques: Clone Assay, Plasmid Preparation, Selection, Marker

    Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to Dynabeads/M-280 streptavidin and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.

    Journal: Journal of Virology

    Article Title: Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor

    doi: 10.1128/JVI.02413-16

    Figure Lengend Snippet: Mutational analyses of the MCAT motif and MCAT-like sequences by reporter and in vitro DNA pulldown assays. (A) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of the expression plasmid for TEAD4 (pCMV-HA-TEAD4) and the Renilla luciferase plasmid. Two days after transfection, the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. The levels of luciferase activity are presented as fold increase compared with that obtained from cells transfected with pA3B−200/+45 without the TEAD4 expression plasmid. The data are the averages of three independent experiments, with the error bars representing standard deviations. (B) Schematic representation of the biotinylated DNA probes used in DNA pulldown assays. The positions and nucleotide sequences of MCAT-like 1 and 2 and MCAT are shown. The mutated nucleotides are underlined, and the nucleotide sequences in the mutated probes are denoted by lowercase letters. (C to F) The indicated biotinylated DNA probes were coupled to Dynabeads/M-280 streptavidin and incubated with the nuclear extract from NIKS-E6; 1/10 or 1/20 volume of input (Input) and entire precipitated fractions were analyzed by immunoblotting using anti-pan-TEAD antibody. (E) Unlabeled oligonucleotide competitors (lines A to D) indicated by the solid lines under the nucleotide sequence of the −141/−96 probe were added to the reaction mixtures, and inhibition of TEADs binding to the −141/−96 probe was examined. The MCAT-like sequences 1 and 2 are boxed, and nucleotides that match the typical MCAT motif are underlined. (G) HEK293 cells were transfected with the indicated firefly luciferase reporter plasmid, together with the indicated amounts of pCMV-HA-TEAD4 and the Renilla luciferase plasmid, and the luciferase activity was measured as described for panel A.

    Article Snippet: The biotinylated DNA probes (12.5 pmol) were coupled to 200 μg of Dynabeads/M-280 streptavidin (Dynal Biotech, Oslo, Norway) at room temperature for 30 min in a coupling buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 M NaCl).

    Techniques: In Vitro, Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Incubation, Sequencing, Inhibition, Binding Assay

    Map of pGADT7-DEST. This plasmid was modified from pGADT7-Rec (Clontech Laboratories); the SMART III and CDS III sequences were replaced by attR1 and attR2, respectively. Following recombination catalysis using the Gateway ® LR Clonase ™ II enzyme mix (Invitrogen), attR1-Cm-ccdB-attR2 was replaced by attB1-cDNA insert-attB2.

    Journal: PLoS ONE

    Article Title: Envelope Proteins of White Spot Syndrome Virus (WSSV) Interact with Litopenaeus vannamei Peritrophin-Like Protein (LvPT)

    doi: 10.1371/journal.pone.0144922

    Figure Lengend Snippet: Map of pGADT7-DEST. This plasmid was modified from pGADT7-Rec (Clontech Laboratories); the SMART III and CDS III sequences were replaced by attR1 and attR2, respectively. Following recombination catalysis using the Gateway ® LR Clonase ™ II enzyme mix (Invitrogen), attR1-Cm-ccdB-attR2 was replaced by attB1-cDNA insert-attB2.

    Article Snippet: The cDNA library plasmid was recombined with modified pGADT7-Rec (or pGADT7- DEST; ) using the Gateway® LR Clonase™ II enzyme mix (Invitrogen).

    Techniques: Plasmid Preparation, Modification

    Tandem affinity purification (TAP) and proteomic analysis of TbMCU complex. (A) Diagram showing positions of tags. TEV, tobacco etch virus protease cleavage site; MH, Myc-His; CBP, calmodulin-binding peptide; Prot A, protein A domain. (B) Scheme of the TAP method used for isolation of TAP-tagged TbMCU complex. (C) Western blot analyses of tetracycline-inducible TAP-tagged TbMCU-TAP overexpressed in PCF trypanosomes against the CBP, MYC, and TbMCU antibodies. One additional band at approximately 40 kDa was detected, possibly because of degradation of the TAP-tagged TbMCU protein (arrows) by the AcTEV protease or an alternative translation termination at the TEV cleavage site. When anti-TbMCU antibody was used, both the TAP-tagged protein and the endogenous copy were detected. The blot was stripped and reincubated with antitubulin (α-Tub) antibody as a loading control. (D) SDS-PAGE of purified TbMCU complex (left, silver stained [SS]; right, immunoblotted with anti-TbMCU). M, PageRuler unstained protein ladder.

    Journal: mBio

    Article Title: The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans

    doi: 10.1128/mBio.00268-20

    Figure Lengend Snippet: Tandem affinity purification (TAP) and proteomic analysis of TbMCU complex. (A) Diagram showing positions of tags. TEV, tobacco etch virus protease cleavage site; MH, Myc-His; CBP, calmodulin-binding peptide; Prot A, protein A domain. (B) Scheme of the TAP method used for isolation of TAP-tagged TbMCU complex. (C) Western blot analyses of tetracycline-inducible TAP-tagged TbMCU-TAP overexpressed in PCF trypanosomes against the CBP, MYC, and TbMCU antibodies. One additional band at approximately 40 kDa was detected, possibly because of degradation of the TAP-tagged TbMCU protein (arrows) by the AcTEV protease or an alternative translation termination at the TEV cleavage site. When anti-TbMCU antibody was used, both the TAP-tagged protein and the endogenous copy were detected. The blot was stripped and reincubated with antitubulin (α-Tub) antibody as a loading control. (D) SDS-PAGE of purified TbMCU complex (left, silver stained [SS]; right, immunoblotted with anti-TbMCU). M, PageRuler unstained protein ladder.

    Article Snippet: The protein-bead mix was resuspended in 1 ml of TEV buffer containing 10 μl of AcTEV protease (Invitrogen; 10 units/μl) and then incubated at 16°C for 2 h with constant mixing.

    Techniques: Affinity Purification, Binding Assay, Isolation, Western Blot, SDS Page, Purification, Staining

    vRNA binding by RIG-I. GFP-RIG-I expressed by 293T cells was coupled to protein G Sepharose beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H 2 O was used as negative control.

    Journal: PLoS ONE

    Article Title: Processing of Genome 5? Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

    doi: 10.1371/journal.pone.0002032

    Figure Lengend Snippet: vRNA binding by RIG-I. GFP-RIG-I expressed by 293T cells was coupled to protein G Sepharose beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H 2 O was used as negative control.

    Article Snippet: In parallel, 20 µl of a 50% slurry of protein G Sepharose in RIPA buffer were preadsorbed with 5 µl polyclonal rabbit anti-GFP antiserum (Invitrogen) and incubated for 2 h at 4°C.

    Techniques: Binding Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Analysis of the capability of Sen1 variants to terminate transcription in vitro . ( A ) Scheme of an in vitro transcription termination assay. A schematic of a ternary EC based on previous biochemical and structural analyses ( 47 , 48 ) is shown on the top. Promoter-independent assembly of ECs is performed using a 9 nt RNA:DNA hybrid that occupies the RNAPII catalytic center. Ternary ECs are attached to streptavidin beads via the 5΄ biotin of the non-template strand allowing subsequent separation of bead-associated (B) and supernatant (S) fractions. The RNA is fluorescently labeled with FAM at the 5΄-end. The transcription template contains a G-less cassette followed by a G-stretch in the non-template strand. After adding an ATP, UTP, CTP mix, the RNAPII transcribes until it encounters the G-rich sequence. Sen1 provokes dissociation of ECs paused at the G-rich stretch and therefore the release of RNAPII and associated transcripts to the supernatant. ( B ) Denaturing PAGE analysis of RNAs from a representative IVTer assay in the absence and in the presence of Sen1 proteins. ( C ) Quantification of the fraction of transcripts released from ECs stalled at the G-stretch as a measure of the termination efficiency. Values represent the average and standard deviation of three independent experiments. The p-value associated with a t -test (p) is indicated.

    Journal: Nucleic Acids Research

    Article Title: Biochemical characterization of the helicase Sen1 provides new insights into the mechanisms of non-coding transcription termination

    doi: 10.1093/nar/gkw1230

    Figure Lengend Snippet: Analysis of the capability of Sen1 variants to terminate transcription in vitro . ( A ) Scheme of an in vitro transcription termination assay. A schematic of a ternary EC based on previous biochemical and structural analyses ( 47 , 48 ) is shown on the top. Promoter-independent assembly of ECs is performed using a 9 nt RNA:DNA hybrid that occupies the RNAPII catalytic center. Ternary ECs are attached to streptavidin beads via the 5΄ biotin of the non-template strand allowing subsequent separation of bead-associated (B) and supernatant (S) fractions. The RNA is fluorescently labeled with FAM at the 5΄-end. The transcription template contains a G-less cassette followed by a G-stretch in the non-template strand. After adding an ATP, UTP, CTP mix, the RNAPII transcribes until it encounters the G-rich sequence. Sen1 provokes dissociation of ECs paused at the G-rich stretch and therefore the release of RNAPII and associated transcripts to the supernatant. ( B ) Denaturing PAGE analysis of RNAs from a representative IVTer assay in the absence and in the presence of Sen1 proteins. ( C ) Quantification of the fraction of transcripts released from ECs stalled at the G-stretch as a measure of the termination efficiency. Values represent the average and standard deviation of three independent experiments. The p-value associated with a t -test (p) is indicated.

    Article Snippet: The ternary ECs were then immobilized on streptavidin beads (Dynabeads MyOne Streptavidin T1 from Invitrogen) and washed with transcription buffer (TB) containing 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 8 mM MgCl2 , 10 μM ZnCl2 , 10% glycerol and 2 mM DTT; then with TB/0.1% Triton, TB/0.5 M NaCl and finally TB.

    Techniques: In Vitro, Labeling, Sequencing, Polyacrylamide Gel Electrophoresis, Standard Deviation

    PROP1 is a regulator of genes involved in EMT. ( A ) HOMER analysis of new motif enrichment within PROP1 peaks. ( B ) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. ( C ) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. ( D ) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p

    Journal: eLife

    Article Title: PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells

    doi: 10.7554/eLife.14470

    Figure Lengend Snippet: PROP1 is a regulator of genes involved in EMT. ( A ) HOMER analysis of new motif enrichment within PROP1 peaks. ( B ) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. ( C ) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. ( D ) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p

    Article Snippet: After sonication, 10 µg of soluble chromatin was incubated with streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) at 4°C overnight.

    Techniques: Chromatin Immunoprecipitation

    Characterization of Prop1-biotag transgenic mice. ( A ) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). ( B ) Body weight at P21. Prop1 -/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p

    Journal: eLife

    Article Title: PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells

    doi: 10.7554/eLife.14470

    Figure Lengend Snippet: Characterization of Prop1-biotag transgenic mice. ( A ) Pituitary paraffin sections were prepared from e12.5 embryos of the genotype indicated and immunohistochemistry was performed. A polyclonal antibody against PROP1 detects the PROP1 protein in the pituitary primordium - Rathke’s Pouch (upper panel). The transgenic PROP1-biotag protein can be detected with fluorescein-conjugated streptavidin only when is co-expressed with BirA (lower panel). ( B ) Body weight at P21. Prop1 -/- mice are dwarfed, but the presence of the Prop1-biotag transgene alone or co-expressed with BirA rescues the dwarf phenotype. The mean of body weight of mice of each genotype is graphed. Error bars represent the standard deviation. (n = 25–40) ANOVA *p

    Article Snippet: After sonication, 10 µg of soluble chromatin was incubated with streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) at 4°C overnight.

    Techniques: Transgenic Assay, Mouse Assay, Immunohistochemistry, Standard Deviation

    Improvement of the sybody selection procedure. ( A ) Three rounds of ribosome display using the same type of magnetic beads for target immobilization (Dynabeads Myone Streptavidin T1) failed to generate sybodies against ABC transporter TM287/288. Pool enrichment against TM287/288 compared to negative control AcrB was poor. No positive ELISA hits were identified. ( B ) Sybody selections against TM287/288 were performed applying one round of ribosome display followed by two rounds of phage display using Dynabeads Myone Streptavidin T1 for target immobilization. The pool was enriched approximately 30 fold and a few positive ELISA hits were found. Purification of identified sybodies failed. ( C ) Sybody selections against ABC transporter IrtAB, a homologue of TM287/288 sharing a sequence identity of 27%, was performed as in ( B ), but using different immobilization chemistries (Dynabeads Myone Streptavidin T1 for ribosome display, Maxisorp microtiter plates for the first phage display round and Dynabeads Myone Streptavidin C1 for the second phage display round) to suppress accumulation of background binders. Strong enrichment was observed and a high number of positive ELISA hits were identified. Only 27% of positive ELISA hits were unique sybodies with moderate affinities. ( D ) Final optimized sybody selection protocol as described in the materials and methods section. Diversity bottlenecks were removed by using Taq DNA polymerase for cDNA amplification and increasing the working volume of the first phage display round. An off-rate selection step was introduced in the second phage display round. Enrichment and number of ELISA hits was similar to the selection shown in ( C ). The number of unique ELISA hits increased to 83% and high affinity binders were obtained. The binders obtained in ( D ) against TM287/288 are described in detail in main Figures 3 and 4 .

    Journal: eLife

    Article Title: Synthetic single domain antibodies for the conformational trapping of membrane proteins

    doi: 10.7554/eLife.34317

    Figure Lengend Snippet: Improvement of the sybody selection procedure. ( A ) Three rounds of ribosome display using the same type of magnetic beads for target immobilization (Dynabeads Myone Streptavidin T1) failed to generate sybodies against ABC transporter TM287/288. Pool enrichment against TM287/288 compared to negative control AcrB was poor. No positive ELISA hits were identified. ( B ) Sybody selections against TM287/288 were performed applying one round of ribosome display followed by two rounds of phage display using Dynabeads Myone Streptavidin T1 for target immobilization. The pool was enriched approximately 30 fold and a few positive ELISA hits were found. Purification of identified sybodies failed. ( C ) Sybody selections against ABC transporter IrtAB, a homologue of TM287/288 sharing a sequence identity of 27%, was performed as in ( B ), but using different immobilization chemistries (Dynabeads Myone Streptavidin T1 for ribosome display, Maxisorp microtiter plates for the first phage display round and Dynabeads Myone Streptavidin C1 for the second phage display round) to suppress accumulation of background binders. Strong enrichment was observed and a high number of positive ELISA hits were identified. Only 27% of positive ELISA hits were unique sybodies with moderate affinities. ( D ) Final optimized sybody selection protocol as described in the materials and methods section. Diversity bottlenecks were removed by using Taq DNA polymerase for cDNA amplification and increasing the working volume of the first phage display round. An off-rate selection step was introduced in the second phage display round. Enrichment and number of ELISA hits was similar to the selection shown in ( C ). The number of unique ELISA hits increased to 83% and high affinity binders were obtained. The binders obtained in ( D ) against TM287/288 are described in detail in main Figures 3 and 4 .

    Article Snippet: 10 µl Dynabeads MyOne Streptavidin T1 (Life Technologies) were washed 3 times with 500 µl WTB and blocked with 500 µl WTB 0.5% BSA for 1 hr followed by 3 washes 500 µl of WTB-T-BSA (0.05% Tween 20, 0.5% BSA).

    Techniques: Selection, Magnetic Beads, Negative Control, Enzyme-linked Immunosorbent Assay, Purification, Sequencing, Amplification

    Features of Drosophila U7 snRNA. ( A ) The sequence and a possible secondary structure of 71-nt Drosophila U7 snRNA. The putative Sm-binding site is underlined. ( B ) Comparison of the Sm-binding site from four previously identified U7 snRNAs and from Drosophila U7 snRNA. The consensus sequence for the previously identified U7 snRNAs is shown and the invariant nucleotides in the U7 snRNA of all four species are boxed. The arrowhead indicates the nucleotide in Drosophila Sm-binding site that departs from the consensus. ( C ) RNA extracted from a Kc nuclear extract (lane 1) was incubated with either a nonspecific mAb (lane 2) or αTMG Ab (lane 3), and the RNA was recovered from protein G-Sepharose and analyzed by Northern blotting. A small amount of 32 P-labeled 86-nt RNA (asterisk) was added to each sample before ethanol precipitation to control for the efficiency of RNA recovery.

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

    Article Title: Cloning and characterization of the Drosophila U7 small nuclear RNA

    doi: 10.1073/pnas.1533509100

    Figure Lengend Snippet: Features of Drosophila U7 snRNA. ( A ) The sequence and a possible secondary structure of 71-nt Drosophila U7 snRNA. The putative Sm-binding site is underlined. ( B ) Comparison of the Sm-binding site from four previously identified U7 snRNAs and from Drosophila U7 snRNA. The consensus sequence for the previously identified U7 snRNAs is shown and the invariant nucleotides in the U7 snRNA of all four species are boxed. The arrowhead indicates the nucleotide in Drosophila Sm-binding site that departs from the consensus. ( C ) RNA extracted from a Kc nuclear extract (lane 1) was incubated with either a nonspecific mAb (lane 2) or αTMG Ab (lane 3), and the RNA was recovered from protein G-Sepharose and analyzed by Northern blotting. A small amount of 32 P-labeled 86-nt RNA (asterisk) was added to each sample before ethanol precipitation to control for the efficiency of RNA recovery.

    Article Snippet: Y12 mouse mAb against Sm proteins (1 ml) was adjusted to 100 mM Tris (pH 8) and rotated with 50 μl of protein G-Sepharose beads (Pierce) for 2 h at 4°C.

    Techniques: Sequencing, Binding Assay, Incubation, Northern Blot, Labeling, Ethanol Precipitation

    Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein G-dynabeads and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.

    Journal: bioRxiv

    Article Title: Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

    doi: 10.1101/2020.07.06.189761

    Figure Lengend Snippet: Pif1 is acetylated both in vivo and in vitro . [A] Top Panel : S. cerevisiae lysates from WT (lane 1), esa1-414 (lane 2), and rpd3Δ (lane 3) backgrounds were immunoprecipitated with anti-acetyl lysine (ac-K) antibody-coated Protein G-dynabeads and immunoblotted with anti-FLAG antibody (1:1000); middle Panel : 10% of input immunoblotted with the anti-FLAG antibody and; bottom panel : PGK-1 antibody (1:10,000). [B] Immunoblot of unmodified Pif1 (lane 1), NuA4 (Esa1) in vitro -acetylated full-length Pif1 (lane 2), unmodified Pif1ΔN, and NuA4 (Esa1) in vitro -acetylated Pif1ΔN probed with anti-Ac-K antibody. [C] Schematic of the full-length Pif1 sequence. The positions of all of the lysine residues in the sequence are denoted with red lines, and all acetylated lysine residues identified by mass spectrometry are denoted with black lines and red filled circles.

    Article Snippet: Specifically, Protein G Dynabeads (Invitrogen 10007D) were incubated with anti-acetyl lysine antibody (CST 9441) with end-over-end rotation for 4 h at 4°C, followed by the addition of 1 mg of cell lysate, which was then rotated overnight at 4°C.

    Techniques: In Vivo, In Vitro, Immunoprecipitation, Sequencing, Mass Spectrometry

    Biotin affinity purification and western blot analysis. Dynabeads MyOne Streptavidin C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,

    Journal: The Biochemical journal

    Article Title: Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif

    doi: 10.1042/BJ20111942

    Figure Lengend Snippet: Biotin affinity purification and western blot analysis. Dynabeads MyOne Streptavidin C1 beads were coated with double-stranded, 5′-biotinylated oligonucleotides and incubated with nuclear extracts from CMT-93 cells. After magnetic separation,

    Article Snippet: Dynabeads MyOne Streptavidin C1 (Invitrogen) were washed three times for 10 min each in washing buffer (10 mM Tris-HCL, pH 7.5, 100 mM NaCl, 2 mM DTT, 0.1% bovine serum albumin).

    Techniques: Affinity Purification, Western Blot, Incubation

    Effect of Foxa1 overexpression or deficiency on A549 cell apoptosis induced by H 2 O 2 . a A549 cells were transfected with pcDNA3.1-Foxa1 and the expression levels of Foxa1 were assessed by Western blotting. Ctrl A549 cells treated with lipofectamine only,

    Journal: Cell Stress & Chaperones

    Article Title: Role of Foxa1 in regulation of bcl2 expression during oxidative-stress-induced apoptosis in A549 type II pneumocytes

    doi: 10.1007/s12192-008-0095-4

    Figure Lengend Snippet: Effect of Foxa1 overexpression or deficiency on A549 cell apoptosis induced by H 2 O 2 . a A549 cells were transfected with pcDNA3.1-Foxa1 and the expression levels of Foxa1 were assessed by Western blotting. Ctrl A549 cells treated with lipofectamine only,

    Article Snippet: Morpholinos were transfected into A549 cells with lipofectamine according to the manufacturer’s instructions (lipofectamine 2000™, Invitrogen) 24 h after plating.

    Techniques: Over Expression, Transfection, Expressing, Western Blot

    Silencing of PLB in CM by amiR155-PLBr and shPLBr at the mRNA and protein level. (A) Dose dependence of RNAi-mediated PLB mRNA silencing detected by Northern blot analysis. CM were transduced with either scAAV6-amiR155-PLBr (amiR155-PLBr), scAAV6-shPLBr (shPLBr) or control AAV vector (amiR155-Con) and the cells were harvested 7 days later. The multiple bands represent alternatively spliced PLB mRNA transcripts of different lengths. (B) Dose dependence of RNAi-mediated PLB silencing at the protein level for transduction conditions described under A. Upper panel : Representative Western blots showing the immunoreactive signals of PLB and GAPDH. Lower panel : Results of the semi-quantitative analysis of the blots. The obtained PLB/GAPDH signal ratio values were normalized to that of non-transduced cells (no add). (C-E) Time dependence of RNAi-mediated PLB silencing. CM were transduced with 25×10 3 vg/c of scAAV6-shPLBr, scAAV6-amiR155-PLBr, scAAV6-shCon, or scAAV6-amiR155-Con as indicated and then analyzed at indicated time points. (C) The PLB mRNA expression levels were determined by qRT-PCR. Quantification of relative PLB mRNA levels was done as described under ( Figure 2B ). (D) Representative Western blot of PLB, SERCA2a and GAPDH. (E) Results of the semi-quantitative Western blotting analysis of four independent experiments. The bars indicate the percentage of PLB ( upper panel ) and SERCA2a ( lower panel ) abundance found in scAAV6-shPLBr (shPLBr) and scAAV6-amiR155-PLBr (amiR155-PLBr) vector transduced cells as compared to respective control vector transduced CM exhibiting PLB and SERCA2a abundance of 100%, respectively. *p

    Journal: PLoS ONE

    Article Title: A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

    doi: 10.1371/journal.pone.0092188

    Figure Lengend Snippet: Silencing of PLB in CM by amiR155-PLBr and shPLBr at the mRNA and protein level. (A) Dose dependence of RNAi-mediated PLB mRNA silencing detected by Northern blot analysis. CM were transduced with either scAAV6-amiR155-PLBr (amiR155-PLBr), scAAV6-shPLBr (shPLBr) or control AAV vector (amiR155-Con) and the cells were harvested 7 days later. The multiple bands represent alternatively spliced PLB mRNA transcripts of different lengths. (B) Dose dependence of RNAi-mediated PLB silencing at the protein level for transduction conditions described under A. Upper panel : Representative Western blots showing the immunoreactive signals of PLB and GAPDH. Lower panel : Results of the semi-quantitative analysis of the blots. The obtained PLB/GAPDH signal ratio values were normalized to that of non-transduced cells (no add). (C-E) Time dependence of RNAi-mediated PLB silencing. CM were transduced with 25×10 3 vg/c of scAAV6-shPLBr, scAAV6-amiR155-PLBr, scAAV6-shCon, or scAAV6-amiR155-Con as indicated and then analyzed at indicated time points. (C) The PLB mRNA expression levels were determined by qRT-PCR. Quantification of relative PLB mRNA levels was done as described under ( Figure 2B ). (D) Representative Western blot of PLB, SERCA2a and GAPDH. (E) Results of the semi-quantitative Western blotting analysis of four independent experiments. The bars indicate the percentage of PLB ( upper panel ) and SERCA2a ( lower panel ) abundance found in scAAV6-shPLBr (shPLBr) and scAAV6-amiR155-PLBr (amiR155-PLBr) vector transduced cells as compared to respective control vector transduced CM exhibiting PLB and SERCA2a abundance of 100%, respectively. *p

    Article Snippet: Plasmids Generation of amiR155 expression cassettes was performed with the Block-iT Pol II miR RNAi Expression Vector Kit according to the recommendations of the supplier (Life Technologies, Invitrogen, Darmstadt, Germany).

    Techniques: Northern Blot, Transduction, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR

    EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different

    Journal: Nature Communications

    Article Title: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii

    doi: 10.1038/s41467-018-03991-6

    Figure Lengend Snippet: EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different

    Article Snippet: To degrade single-stranded DNA and RNA deprived of double-stranded regions, 0.4 μl of S1 nuclease (Thermo Fisher Scientific #EN0321) was added to 20 μl EVsR265 for 30 min at room temperature.

    Techniques: Isolation, Infection, Activation Assay, Incubation