unr mrna sequence  (Thermo Fisher)


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    Structured Review

    Thermo Fisher unr mrna sequence
    SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated <t>mRNA.</t> The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the <t>unr</t> mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).
    Unr Mrna Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells"

    Article Title: Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki968

    SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).
    Figure Legend Snippet: SAABS assay. ( A ) SAABS procedure. A random 8mer oligodeoxynucleotide library (ROL) flanked by two PCR tags is first incubated with biotinylated mRNA. The mRNA bound ODN is then separated from free ODN by binding the biotinylated mRNA to a streptavidin coated Dynabead, which is then separated from the unbound sequence by a magnetic field. The bound sequence is then PCR amplified with S1 and CS2, restricted with NlaIII, concatenated by ligation, cloned in pZErO-1 and sequenced. ( B ) Frequency distribution of the antisense binding sites on the unr mRNA obtained from the SAABS assay. The 8mer sequences were retrieved from the sequenced clones and aligned with the mRNA sequence. Some of the sites identified correspond to sites found by the RT-ROL assay (13 and 46), whereas others were uniquely detected by the SAABS assay and denoted with an S prefix (S1, S3, S5 and S7).

    Techniques Used: Polymerase Chain Reaction, Incubation, Binding Assay, Sequencing, Amplification, Ligation, Clone Assay

    Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.
    Figure Legend Snippet: Dynabead-based dot blot assay to determine relative binding affinity of ODNs. ( A ) Determining the loading capacity of the streptavidin coated Dynabead by titrating 20 µl of bead solution in 40 µl total volume of 0.5 M NaCl with biotinylated radiolabeled unr mRNA. ( B ) Determining the µl of Dynabead bound RNA needed to completely bind 1 pmol of ODN5 in a total volume of 40 µl. ( C and D ) Solutions of RNA were incubated with 1 pmol of ODN [1–54 from RT-ROL assay and 57–68 (S1-S12) from the SAABS assay] and then incubated with 10 µl of Dynabeads and spotted on Nylon membrane. (C) is a photograph of blot showing equal loading of beads. (D) is a radiogram showing relative amounts of retained ODN. ODNs corresponding to circled spots were further studied by quantitative methods.

    Techniques Used: Dot Blot, Binding Assay, Incubation

    2) Product Images from "Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display"

    Article Title: Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2015.06.021

    Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.
    Figure Legend Snippet: Characterization of the binding reactivity of anti-p41_1 and anti-p41_3 antisera. Biotinylated peptides were immobilized onto streptavidin-coated plates. Binding of (A) mAb41 (1.2 μg/ml), (B) anti-p41_1, (C) anti-pA, and (D) anti-p41_3 (1:1000 dilution, n = 3 for (B–D)) to pB and mAb 41-selected peptides (p41_1 to p41_5) were measured by ELISA. Mean values are graphed and error bars represent SEM of technical replicates for (A) mAb41, and biological replicates for antisera (B–D), with each biological replicate having technical duplicates.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.
    Figure Legend Snippet: Binding of selected peptides to the selection mAb41. (A) Binding of synthetic peptides to mAb41 at various concentrations. Biotinylated peptides were added to streptavidin-coated microtiter plates. mAb 41 was applied as the primary antibody in a 10-fold dilution series. HRP-conjugated goat anti-mouse antibody was used as the detection antibody. Results shown are the mean of two replicates performed in one of three independent experiments, each of which showed similar results. (B) Competition for binding to mAb41 between p41_1 and wild type pB. 1 μg biotinylated pB was attached to streptavidin-coated plates. 12 ng of mAb41in 100 μl PBS containing 5% milk was added in the presence of increasing amount of p41_1 as indicated. The bound mAb41 was detected using an HRP-conjugated goat anti-mouse antibody. An unrelated peptide from the mRNA display library was used as a negative control. Mean values are graphed and error bars represent SEM of sample replicates. (C) Measurement of binding affinity of selected peptides to mAb41 by Octet RED. Biotinylated peptides were immobilized on Streptavidin biosensors (Fortebio) and Fabs of mAb41 were used as analyte in a 2-fold dilution series ranging from 125 nM to 7.8 nM. Sensorgram data for peptide p41_1 are shown. (D) Binding constants of mAb41-selected peptides to mAb41 Fabs were obtained by fitting sensorgrams (blue) with a 1:1 model (red) using ForteBio Data Analysis Software. p41_2 is not included due to unsatisfactory curve fitting.

    Techniques Used: Binding Assay, Selection, Negative Control, Software

    3) Product Images from "Dynamic m6A mRNA methylation directs translational control of heat shock response"

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response

    Journal: Nature

    doi: 10.1038/nature15377

    m 6 A modification promotes cap-independent translation a, Fluc reporter mRNAs with or without 5′UTR was synthesized in the absence or presence of m 6 A. The transfected MEFs were incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m.; n=3, biological replicates. b , Fluc reporter mRNAs with or without Hsp70 5′UTR was synthesized in the absence of presence of m 6 A, followed by addition of a non-functional cap analog A ppp G. Fluc activity in transfected MEF cells was recorded using real-time luminometry. c , Constructs expressing Fluc reporter bearing 5′UTR from Hsc70 or Hsp105 are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: m 6 A modification promotes cap-independent translation a, Fluc reporter mRNAs with or without 5′UTR was synthesized in the absence or presence of m 6 A. The transfected MEFs were incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m.; n=3, biological replicates. b , Fluc reporter mRNAs with or without Hsp70 5′UTR was synthesized in the absence of presence of m 6 A, followed by addition of a non-functional cap analog A ppp G. Fluc activity in transfected MEF cells was recorded using real-time luminometry. c , Constructs expressing Fluc reporter bearing 5′UTR from Hsc70 or Hsp105 are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Synthesized, Transfection, Incubation, Real-time Polymerase Chain Reaction, Functional Assay, Activity Assay, Construct, Expressing

    mRNA stability and induction in response to heat shock stress a, Effects of heat shock stress on mRNA stability. MEF cells without heat shock stress (No HS), immediately after heat shock stress (42°C, 1 h) (Post HS 0h), or 2 h recovery at 37°C (Post HS 2h) were subject to further incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m. n=3. b , MEF cells were collected at indicated times after heat shock stress (42°C, 1 h) followed by RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m. n=3, biological replicates. c , HSF1 WT and KO cells were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify transcripts encoding Hsp70 and YTHDF2. Relative levels of transcripts are normalized to β-actin. Error bars, mean ± s.e.m. *, p
    Figure Legend Snippet: mRNA stability and induction in response to heat shock stress a, Effects of heat shock stress on mRNA stability. MEF cells without heat shock stress (No HS), immediately after heat shock stress (42°C, 1 h) (Post HS 0h), or 2 h recovery at 37°C (Post HS 2h) were subject to further incubation in the presence of 5 μg/ml ActD. At the indicated times, mRNA levels were determined by qPCR. Error bars, mean ± s.e.m. n=3. b , MEF cells were collected at indicated times after heat shock stress (42°C, 1 h) followed by RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m. n=3, biological replicates. c , HSF1 WT and KO cells were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify transcripts encoding Hsp70 and YTHDF2. Relative levels of transcripts are normalized to β-actin. Error bars, mean ± s.e.m. *, p

    Techniques Used: Incubation, Real-time Polymerase Chain Reaction, RNA Extraction

    YTHDF2 knockdown does not affect Hsp70 transcription after stress MEF cells with or without YTHDF2 knockdown were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify Hsp70 mRNA levels. Error bars, mean ± s.e.m.; n=3, biological replicates.
    Figure Legend Snippet: YTHDF2 knockdown does not affect Hsp70 transcription after stress MEF cells with or without YTHDF2 knockdown were subject to heat shock stress (42°C, 1 h) followed by recovery at 37°C for various times. Real-time PCR was conducted to quantify Hsp70 mRNA levels. Error bars, mean ± s.e.m.; n=3, biological replicates.

    Techniques Used: Real-time Polymerase Chain Reaction

    Selective 5′UTR m 6 A modification mediates cap-independent translation a, MEF cells transfected with Fluc mRNA reporters were subject to heat shock treatment and the Fluc activity was measured by real-time luminometry. Fluc activities were quantified and normalized to the one containing normal As. b , Constructs expressing Fluc reporter with Hsp70 5′UTR or the one with A103C mutation are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A without stress. c , Fluc mRNAs bearing Hsp70 5′UTR with a single m 6 A site were constructed using sequential splint ligation. After in vitro translation in rabbit reticulate lysates, Fluc activities were quantified and normalized to the control lacking m 6 A. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: Selective 5′UTR m 6 A modification mediates cap-independent translation a, MEF cells transfected with Fluc mRNA reporters were subject to heat shock treatment and the Fluc activity was measured by real-time luminometry. Fluc activities were quantified and normalized to the one containing normal As. b , Constructs expressing Fluc reporter with Hsp70 5′UTR or the one with A103C mutation are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A without stress. c , Fluc mRNAs bearing Hsp70 5′UTR with a single m 6 A site were constructed using sequential splint ligation. After in vitro translation in rabbit reticulate lysates, Fluc activities were quantified and normalized to the control lacking m 6 A. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Transfection, Activity Assay, Construct, Expressing, Mutagenesis, Ligation, In Vitro

    4) Product Images from "Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3"

    Article Title: Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011941

    Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.
    Figure Legend Snippet: Binding specificities of anti-VEGF-C scFv. (A) ELISA screening of random clones obtained after 2 or 3 rounds of panning against ΔNΔC-VEGF-C. (B) ELISA analysis of representative anti-VEGF-C scFv clones for the 4 different amino acid sequences obtained. Maxisorp or streptavidin-precoated (SA) plates were coated with his-tagged human ΔNΔC-VEGF-C derived from P. pastoris or biotinylated his-tagged human ΔNΔC-VEGF-C from mammalian cells or P. pastoris , respectively. Control surfaces were left untreated. Antibody fragments and control antibodies were subsequently added and the ELISA was developed as described in Materials and Methods. (C) Cross-reactivity tested by ELISA. Human ΔNΔC-VEGF-C orΔNΔC-VEGF-D (both from mammalian cells) were coated on a maxisorp plate. Anti-VEGF-C scFv clone VC2 or a negative control (PBS only) was added and the ELISA was developed as described in Materials and Methods. (D) BIAcore profiles from the 4 different anti-VEGF-C scFv clones. Different concentrations of protein-A purified scFv were injected on a streptavidin-precoated sensorchip coated with ca. 2000 RU biotinylated mammalian cell-derived ΔNΔC-VEGF-C.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Derivative Assay, Negative Control, Purification, Injection

    Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.
    Figure Legend Snippet: Affinity matured anti-VEGF-C scFvs possess a higher affinity. (A, B) ELISA analysis of bacterial supernatant from randomly picked affinity matured clones after 1 to 3 rounds of selection on biotinylated (A) P. pastoris -derived or (B) mammalian cell-derived ΔNΔC-VEGF-C. (C) BIAcore profiles of monomeric affinity matured anti-VEGF-C scFvs. Monomeric fractions of protein-A purified scFv were prepared by FPLC and injected as 2-fold dilution series on a streptavidin-sensorchip coated with 2000 RU biotinylated ΔNΔC-VEGF-C derived from mammalian cells.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Clone Assay, Selection, Derivative Assay, Purification, Fast Protein Liquid Chromatography, Injection

    5) Product Images from "The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and ?-Lactams in Staphylococcus aureus ▿"

    Article Title: The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and ?-Lactams in Staphylococcus aureus ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01819-06

    (A) Gel mobility shift analyses of the interactions of the crude cell extracts (CE) from ISP794 and QT1 and the purified NorG protein with the biotinylated 150-bp norA promoter fragment. Competing unlabeled herring sperm DNA (nonspecific) and norA promoter DNA (specific) were used to determine the specificity of promoter binding. The amount of labeled DNA used was 2 ng per reaction. The amount of protein used was 50 ng per reaction for the NorG protein and 100 ng per reaction for the crude cell extracts. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and the purified NorG protein with the biotinylated 150-bp norB P1 and P2 promoter fragments. (C) Schematic representation of the positions of norB and the three adjacent ORFs on the S. aureus ). The two putative promoters and the putative rho -dependent terminator are indicated.
    Figure Legend Snippet: (A) Gel mobility shift analyses of the interactions of the crude cell extracts (CE) from ISP794 and QT1 and the purified NorG protein with the biotinylated 150-bp norA promoter fragment. Competing unlabeled herring sperm DNA (nonspecific) and norA promoter DNA (specific) were used to determine the specificity of promoter binding. The amount of labeled DNA used was 2 ng per reaction. The amount of protein used was 50 ng per reaction for the NorG protein and 100 ng per reaction for the crude cell extracts. (B) Gel mobility shift analyses of the interactions of the crude cell extracts from ISP794 and the purified NorG protein with the biotinylated 150-bp norB P1 and P2 promoter fragments. (C) Schematic representation of the positions of norB and the three adjacent ORFs on the S. aureus ). The two putative promoters and the putative rho -dependent terminator are indicated.

    Techniques Used: Mobility Shift, Purification, Binding Assay, Labeling

    6) Product Images from "Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry"

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006766

    Phosphatidylserines persist at the outer leaflet when cells are exposed to viruses deleted in glycoprotein L. (A). CaSki cells were mock-infected or infected with HSV-2(G), complemented or non-complemented ΔgL-2 (ΔgL-2 +/- or ΔgL-2 -/- ), KOS, and complemented or non-complemented ΔgL-1 (ΔgL-1 +/- or ΔgL-1 -/- ) viruses (MOI ~ 1 pfu/cell) and then fixed and stained 30 min or 4 h pi for PLSCR1 (green) (rabbit polyclonal anti-PLSCR1 and secondary Alexa 488 Ab), PtdS (red) (annexin-V Alexa 555) or Akt (green) (rabbit polyclonal and secondary Alexa 488 Abs). Representative images from 2–4 experiments are shown). (B). The percentages of cells with detectable PLSCR1, PtdS and Akt were determined by counting 80–100 cells in two independent experiments; results are means + SEM (* p
    Figure Legend Snippet: Phosphatidylserines persist at the outer leaflet when cells are exposed to viruses deleted in glycoprotein L. (A). CaSki cells were mock-infected or infected with HSV-2(G), complemented or non-complemented ΔgL-2 (ΔgL-2 +/- or ΔgL-2 -/- ), KOS, and complemented or non-complemented ΔgL-1 (ΔgL-1 +/- or ΔgL-1 -/- ) viruses (MOI ~ 1 pfu/cell) and then fixed and stained 30 min or 4 h pi for PLSCR1 (green) (rabbit polyclonal anti-PLSCR1 and secondary Alexa 488 Ab), PtdS (red) (annexin-V Alexa 555) or Akt (green) (rabbit polyclonal and secondary Alexa 488 Abs). Representative images from 2–4 experiments are shown). (B). The percentages of cells with detectable PLSCR1, PtdS and Akt were determined by counting 80–100 cells in two independent experiments; results are means + SEM (* p

    Techniques Used: Infection, Staining

    Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p
    Figure Legend Snippet: Phospholipid scramblase blockade prevents HSV-induced Akt phosphorylation, Ca 2+ release and viral entry. (A) CaSki cells were transfected with siControl or siPLSCR1 and 72 h post-transfection were infected with purified HSV-2(G) (10 pfu/cell). Western blots of cell lysates harvested at the indicated times post-infection were probed with anti- pS 473 -Akt and then stripped and probed with anti-total Akt and anti-PLSCR1 antibodies. Representative blots from 2 independent experiments are shown. Images were scanned and pAkt as a percentage of total Akt was calculated (mean+ SEM). (B) CaSki cells were transfected with siRNA as in (A), loaded with Fura-2 and then infected with purified HSV-2(G) (5 pfu/cell), HSV-1(KOS) or mock-infected and the kinetics of calcium response monitored over 60 minutes. (C) The mean calcium released over 1 h was calculated from 4 wells in 3 independent experiments, each containing 5 x10 4 cells. As additional controls, siRNA transfected cells were treated with ionomycin. The asterisks indicate significant differences in Ca 2+ concentration relative to mock-infected controls (*, p

    Techniques Used: Transfection, Infection, Purification, Western Blot, Concentration Assay

    Phospholipid scramblase blockade reduces HSV entry and viral plaque formation: (A) CaSki cells were pretreated with 0.5% DMSO or 5 μM R5421 for 15 minutes and then infected at 37° with HSV-2(G) (10 pfu/cell) for 30 minutes. Cells were fixed and stained with fluorescently-conjugated antibodies to PtdS; nuclei were stained with DAPI. Representative images from 2 independent experiments are shown. (B) Vk2E6/E7 (HSV) or Vero (VSV) cells were pretreated with R5421 or DMSO control as in (A) and infected with HSV-2(333ZAG), HSV-1(VP26GFP) or VSV-GFP (0.1 pfu/cell). The percentage of GFP-positive cells was quantified 16 h post-infection by counting 400–500 cells in total from 4 random fields in 2 independent experiments. Results are presented as mean + SEM and the asterisks indicate p
    Figure Legend Snippet: Phospholipid scramblase blockade reduces HSV entry and viral plaque formation: (A) CaSki cells were pretreated with 0.5% DMSO or 5 μM R5421 for 15 minutes and then infected at 37° with HSV-2(G) (10 pfu/cell) for 30 minutes. Cells were fixed and stained with fluorescently-conjugated antibodies to PtdS; nuclei were stained with DAPI. Representative images from 2 independent experiments are shown. (B) Vk2E6/E7 (HSV) or Vero (VSV) cells were pretreated with R5421 or DMSO control as in (A) and infected with HSV-2(333ZAG), HSV-1(VP26GFP) or VSV-GFP (0.1 pfu/cell). The percentage of GFP-positive cells was quantified 16 h post-infection by counting 400–500 cells in total from 4 random fields in 2 independent experiments. Results are presented as mean + SEM and the asterisks indicate p

    Techniques Used: Infection, Staining

    Phospholipid scramblase is required for phosphatidylserine and Akt relocalization. (A) CaSki cells were mock-infected or infected with HSV-2(G) and 15 minutes, 30 minutes, 1 hour and 4 hours post-infection, cell lysates were harvested. Lysates were incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein A-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine (PY20) or mouse anti-PLSCR1 mAb. The blot is representative of results obtained in 2 independent experiments. (B). CaSki, VK2E6/E7 or HaCAT cells were transfected with siRNA targeting PLSCR1 or a control siRNA (siCtrl) and protein expression was evaluated by Western blot probing for PLSCR1 (rabbit anti-PLSCR1) and β-actin (mouse monoclonal). Blot is representative of results obtained in 3 independent experiments. (C) CaSki or HaCAT cells were transfected with siControl (siCtrl) or siPLSCR1 RNA and 72 h post-transfection, plasma membranes were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (red) and then synchronously infected with HSV-1(KOS), HSV-2(G), or mock-infected (4 hours at 4°C, washed, and then shifted to 37°C for 15 min and treated with low pH citrate buffer). The cells were then fixed and nuclei were stained blue with Hoechst, and phosphatidylserines (PtdS) or PLSCR1 stained green with respective primary murine and secondary Alexa 488-conjugated secondary antibodies. Images are representative of results obtained from 2–3 independent experiments. (D). CaSki cells were transfected with the indicated siRNA and then synchronously infected with HSV-2(G), fixed with or without Triton X permeabilization and stained with fluorescently-conjugated antibodies to Akt (red) and PLSCR1 (green); nuclei were stained blue with DAPI. Representative 3-D images from 3 independent experiments are shown; bars = 10μm.
    Figure Legend Snippet: Phospholipid scramblase is required for phosphatidylserine and Akt relocalization. (A) CaSki cells were mock-infected or infected with HSV-2(G) and 15 minutes, 30 minutes, 1 hour and 4 hours post-infection, cell lysates were harvested. Lysates were incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein A-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine (PY20) or mouse anti-PLSCR1 mAb. The blot is representative of results obtained in 2 independent experiments. (B). CaSki, VK2E6/E7 or HaCAT cells were transfected with siRNA targeting PLSCR1 or a control siRNA (siCtrl) and protein expression was evaluated by Western blot probing for PLSCR1 (rabbit anti-PLSCR1) and β-actin (mouse monoclonal). Blot is representative of results obtained in 3 independent experiments. (C) CaSki or HaCAT cells were transfected with siControl (siCtrl) or siPLSCR1 RNA and 72 h post-transfection, plasma membranes were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (red) and then synchronously infected with HSV-1(KOS), HSV-2(G), or mock-infected (4 hours at 4°C, washed, and then shifted to 37°C for 15 min and treated with low pH citrate buffer). The cells were then fixed and nuclei were stained blue with Hoechst, and phosphatidylserines (PtdS) or PLSCR1 stained green with respective primary murine and secondary Alexa 488-conjugated secondary antibodies. Images are representative of results obtained from 2–3 independent experiments. (D). CaSki cells were transfected with the indicated siRNA and then synchronously infected with HSV-2(G), fixed with or without Triton X permeabilization and stained with fluorescently-conjugated antibodies to Akt (red) and PLSCR1 (green); nuclei were stained blue with DAPI. Representative 3-D images from 3 independent experiments are shown; bars = 10μm.

    Techniques Used: Infection, Incubation, Western Blot, Transfection, Expressing, Staining

    Viral binding to heparan sulfate and gD-nectin engagement precede and are required for activation of phospholipid scramblase. (A). CaSki or HaCATcells were transfected with siControl or siPLSCR1 and 72 h later were exposed to indicated multiplicities of infection (MOI) of HSV-2(G) for 4 hours at 4°C. The cells were then washed extensively and Western blots of cell lysates were probed with a mAb to gD as a marker of cell-bound virus, mAb for α-tubulin as a control for cell loading and rabbit anti-PLSCR1 as a probe for silencing. The blot is representative of results obtained in 2 independent experiments. (B). CaSki or HaCAT cells were transfected with control (Ctrl) or PLSCR1 siRNA and then infected with envelope-labeled (red) HSV-1VP26GFP (MOI 5 pfu/cell) for 4 hours at 4°C to detect binding or incubated for an additional 4 h at 37°C to detect viral entry. Results are representative of 2 independent experiments; bar = 10μm. (C). HSV-2(G) (~MOI 5 pfu/cell) was mixed with 100 μg/ml heparin or with 1:100 dilution of mAbs to HSV-2 glycoproteins gB, gD, gC, gL, or gH or control mouse IgG and then applied to CaSki cells that had been prestained with Alexa Fluor 594-conjugated wheat germ agglutinin to detect plasma membranes (red). After a 30-minute incubation, the cells were then washed, fixed and stained without permeabilization. Nuclei were stained blue with Hoechst and phosphatidylserines green with a primary murine and secondary Alexa 488-conjugated secondary antibody. Mock-infected cells are included as a control. Images are representative of results obtained from 3 independent experiments. (D). CaSki cells or HSV-2(G) (calculated to yield ~100 pfu/well) were pretreated (pre-rx) with antibodies to phosphatidylserine (PtdS), gD, Akt, nectin or control mouse IgG for 1 h and then washed 3 times (cells) or diluted 1:100 (virus-antibody mixture). Alternatively, the antibodies were added to cells at the time of viral infection for 1 h (entry). Cells were washed after the 1 h entry period, overlaid with fresh medium and plaques were quantified at 48 hours. Results are presented as pfu/well and are means+ SEM from duplicate wells in 2–3 individual experiments. The asterisks indicate p
    Figure Legend Snippet: Viral binding to heparan sulfate and gD-nectin engagement precede and are required for activation of phospholipid scramblase. (A). CaSki or HaCATcells were transfected with siControl or siPLSCR1 and 72 h later were exposed to indicated multiplicities of infection (MOI) of HSV-2(G) for 4 hours at 4°C. The cells were then washed extensively and Western blots of cell lysates were probed with a mAb to gD as a marker of cell-bound virus, mAb for α-tubulin as a control for cell loading and rabbit anti-PLSCR1 as a probe for silencing. The blot is representative of results obtained in 2 independent experiments. (B). CaSki or HaCAT cells were transfected with control (Ctrl) or PLSCR1 siRNA and then infected with envelope-labeled (red) HSV-1VP26GFP (MOI 5 pfu/cell) for 4 hours at 4°C to detect binding or incubated for an additional 4 h at 37°C to detect viral entry. Results are representative of 2 independent experiments; bar = 10μm. (C). HSV-2(G) (~MOI 5 pfu/cell) was mixed with 100 μg/ml heparin or with 1:100 dilution of mAbs to HSV-2 glycoproteins gB, gD, gC, gL, or gH or control mouse IgG and then applied to CaSki cells that had been prestained with Alexa Fluor 594-conjugated wheat germ agglutinin to detect plasma membranes (red). After a 30-minute incubation, the cells were then washed, fixed and stained without permeabilization. Nuclei were stained blue with Hoechst and phosphatidylserines green with a primary murine and secondary Alexa 488-conjugated secondary antibody. Mock-infected cells are included as a control. Images are representative of results obtained from 3 independent experiments. (D). CaSki cells or HSV-2(G) (calculated to yield ~100 pfu/well) were pretreated (pre-rx) with antibodies to phosphatidylserine (PtdS), gD, Akt, nectin or control mouse IgG for 1 h and then washed 3 times (cells) or diluted 1:100 (virus-antibody mixture). Alternatively, the antibodies were added to cells at the time of viral infection for 1 h (entry). Cells were washed after the 1 h entry period, overlaid with fresh medium and plaques were quantified at 48 hours. Results are presented as pfu/well and are means+ SEM from duplicate wells in 2–3 individual experiments. The asterisks indicate p

    Techniques Used: Binding Assay, Activation Assay, Transfection, Infection, Western Blot, Marker, Labeling, Incubation, Staining

    HSV triggers externalization of phosphatidylserine. (A). CaSki or HaCAT cells were synchronously infected with purified HSV-1(KOS) or HSV-2(G) at MOI of 0.1 or 1 pfu/cell (1 hour at 4°C, washed, then shifted to 37°C for 15 minutes and treated with low pH citrate buffer before being fixed with or without 0.1%Triton X). Nuclei were stained blue with DAPI, phosphatidylserines red with annexin-V conjugated with Alexa555 and flippase (FIC-1) green with rabbit polyclonal anti-flipppase (FIC-1) and secondary anti-rabbit Alexa 488. Images are representative of results obtained from 2 independent experiments; bar = 10μm. (B). HaCAT cells were prestained green with Alexa Fluor 488-conjugated wheat germ agglutinin and then exposed at 37°C to increasing MOI (0–10 pfu/cell) of HSV-2(G) and fixed and stained 30 min pi. Nuclei were stained blue with Hoechst and phosphatidylserines red with a primary murine and secondary Alexa Fluor 555-conjugated secondary antibody. Images are representative of results obtained from 2 independent experiments. Bar = 10μm. (C and D) HaCAT cells were mock-infected or infected with purified HSV-2(G) (MOI 1 or 10 pfu/cell based on Vero cell viral titer) or non-complemented HSV-2ΔgD -/- (relatively equivalent viral particles based on Western blots for VP5) for 20 minutes and then live cells stained for phosphatidylserines using PE-annexin-V. A representative histogram is shown (C) and results from 4 independent experiments are presented as the % annexin-V-positive live cells (mean + SEM) (D). The asterisks indicate significance compared to mock-infected cells by ANOVA; * p
    Figure Legend Snippet: HSV triggers externalization of phosphatidylserine. (A). CaSki or HaCAT cells were synchronously infected with purified HSV-1(KOS) or HSV-2(G) at MOI of 0.1 or 1 pfu/cell (1 hour at 4°C, washed, then shifted to 37°C for 15 minutes and treated with low pH citrate buffer before being fixed with or without 0.1%Triton X). Nuclei were stained blue with DAPI, phosphatidylserines red with annexin-V conjugated with Alexa555 and flippase (FIC-1) green with rabbit polyclonal anti-flipppase (FIC-1) and secondary anti-rabbit Alexa 488. Images are representative of results obtained from 2 independent experiments; bar = 10μm. (B). HaCAT cells were prestained green with Alexa Fluor 488-conjugated wheat germ agglutinin and then exposed at 37°C to increasing MOI (0–10 pfu/cell) of HSV-2(G) and fixed and stained 30 min pi. Nuclei were stained blue with Hoechst and phosphatidylserines red with a primary murine and secondary Alexa Fluor 555-conjugated secondary antibody. Images are representative of results obtained from 2 independent experiments. Bar = 10μm. (C and D) HaCAT cells were mock-infected or infected with purified HSV-2(G) (MOI 1 or 10 pfu/cell based on Vero cell viral titer) or non-complemented HSV-2ΔgD -/- (relatively equivalent viral particles based on Western blots for VP5) for 20 minutes and then live cells stained for phosphatidylserines using PE-annexin-V. A representative histogram is shown (C) and results from 4 independent experiments are presented as the % annexin-V-positive live cells (mean + SEM) (D). The asterisks indicate significance compared to mock-infected cells by ANOVA; * p

    Techniques Used: Infection, Purification, Staining, Western Blot

    Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.
    Figure Legend Snippet: Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.

    Techniques Used: Infection, Staining, Ligation, Western Blot, Purification, Isolation, Expressing, Immunoprecipitation, Negative Control, Incubation

    Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.
    Figure Legend Snippet: Ionomycin activates phospholipid scramblase leading to externalization of phosphatidylserines and Akt. (A). CaSki cells were treated with ionomycin (1μM), HSV-2(G) (MOI 5 pfu/cell) or DMSO (0.1%) for 15 minutes and then fixed and stained with antibodies to PLSCR1(polyclonal rabbit and Alexa 555, red), PtdS (monoclonal antibody and Alexa555, red), or Akt (polyclonal rabbit and secondary Alexa488, green) as indicated. Nuclei were stained blue with DAPI. Images are representative of results obtained from 2–3 independent experiments; bar = 10μm. Five fields were scanned and mean fluorescence intensity (MFI) per cell calculated using ImageJ software (NIH); results are mean +SEM and the asterisks indicate significant differences by ANOVA compared to DMSO control treated cells. (B). CaSki cells were treated as in A for 15 minutes and then cell lysates were harvested, incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein G-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine mAb (PY20) or mouse anti-PLSCR1 Ab. The blot is representative of results obtained in 2 independent experiments. (C). CaSki cells were harvested 5 or 15 minutes after exposure to DMSO, 1μM ionomycin (Iono) or HSV-2(G) (5 pfu/cell). Cell surface proteins were biotinylated and precipitated with streptavidin magnetic beads and the pellet analyzed by immunoblotting with rabbit anti-pAktThr 308 or mouse anti-pAktS 473 Abs. In parallel, cellular lysates were analyzed by immunoblotting for total cellular Akt (rabbit polyclonal Ab). Blots representative of 2 independent experiments are shown. (D). The blots were scanned and fold increase in pAktThr 308 and anti-pAktS 473 relative to DMSO treated cells is depicted (mean+ SEM). (E). HaCAT cells were treated with 1μM ionomycin or DMSO (0.1%) for 10 minutes and then incubated with soluble gL for 30 minutes at 37°C, transferred to ice, and immunoprecipitated (IP) with goat anti-PLSCR1 (left) or with mouse-anti-gL (right). Equivalent volumes of whole cell lysate, supernatant, or pellet (L, S, P) were analyzed by preparing Western blots (WB) and probing with mouse anti-gL, rabbit anti-PLSCR1 or rabbit anti-FIC-1 as a control. The blots are representative of results obtained in 2 independent experiments.

    Techniques Used: Staining, Fluorescence, Software, Incubation, Western Blot, Magnetic Beads, Immunoprecipitation

    Phospholipid scramblase activation requires intracellular calcium and HSV-2 glycoprotein B and D. (A) CaSki cells were treated with 100μM BAPTA or BAPTA-AM and then infected with HSV-2(G) (10 pfu/cell) or mock-infected for 30 minutes. Cells were then fixed and stained with mAb to phosphatidylserines (PtdS) and secondary anti-mouse Alexa-555 (red), rabbit anti-PLSCR1 with Alexa-488 secondary Ab (green), or rabbit anti-Akt with Alexa-488 secondary Ab (green). Nuclei were stained blue with DAPI. Representative extended focus images from 2 independent experiments are shown (bar = 10μm). (B) CaSki cells were exposed to relatively equivalent particle numbers of HSV-2(G) or the indicated non-complemented deletion viruses (particle numbers estimated by comparing VP5 band on Western blots) for 30 minutes and fixed and stained as in panel A. Results are presentative of 4 independent experiments (2 experiments with ΔgL-2 -/- ). (C) The percentage of cells in which PtdS, Akt or PLSCR was detected by immunostaining was calculated by counting 80–100 cells in total from 4 random fields in at least 2 independent experiments (mean + SEM) and the asterisks indicate significant differences relative to HSV-2(G) (***p
    Figure Legend Snippet: Phospholipid scramblase activation requires intracellular calcium and HSV-2 glycoprotein B and D. (A) CaSki cells were treated with 100μM BAPTA or BAPTA-AM and then infected with HSV-2(G) (10 pfu/cell) or mock-infected for 30 minutes. Cells were then fixed and stained with mAb to phosphatidylserines (PtdS) and secondary anti-mouse Alexa-555 (red), rabbit anti-PLSCR1 with Alexa-488 secondary Ab (green), or rabbit anti-Akt with Alexa-488 secondary Ab (green). Nuclei were stained blue with DAPI. Representative extended focus images from 2 independent experiments are shown (bar = 10μm). (B) CaSki cells were exposed to relatively equivalent particle numbers of HSV-2(G) or the indicated non-complemented deletion viruses (particle numbers estimated by comparing VP5 band on Western blots) for 30 minutes and fixed and stained as in panel A. Results are presentative of 4 independent experiments (2 experiments with ΔgL-2 -/- ). (C) The percentage of cells in which PtdS, Akt or PLSCR was detected by immunostaining was calculated by counting 80–100 cells in total from 4 random fields in at least 2 independent experiments (mean + SEM) and the asterisks indicate significant differences relative to HSV-2(G) (***p

    Techniques Used: Activation Assay, Infection, Staining, Western Blot, Immunostaining

    7) Product Images from "Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry"

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006766

    Model of phospholipid scramblase activation during HSV entry. Binding of HSV-1 glycoprotein C or HSV-2 glycoprotein B (Step 1) and engagement of nectin-1 by glycoprotein D (Step 2) trigger the release of a small amount of calcium near the plasma membrane (Step 3) that is sufficient to activate (phosphorylate) phospholipid scramblase (Step 4). This results in flipping of phosphatidylserines (PtdS) from the inner to the outer leaflet of the plasma membrane, which results in externalization of Akt and possibly other inner plasma membrane- associated proteins (Step 5). Akt interacts with gB, which may promote a conformational change in Akt that facilitates its phosphorylation at threonine 308 and serine 473 by yet to be determined kinases (Step 6). Integrinαvβ3 binds to gH (Step 7), and activated Akt and integrin transfer signals to the cell cytoplasm that lead to the generation of inositol triphosphates and release of inositol-triphosphate receptor (IP3R)-regulated Ca 2+ stores (Step 8) culminating in fusion of the viral envelope and plasma membrane and entry of viral capsids (Step 9). PtdS flip back to the inner leaflet of the plasma membrane to restore normal membrane asymmetry in a process that may occur in response to viral entry and may be regulated by gL binding to PLSCR1 and subsequent shift to primarily dephosphorylated PLSCR1. (Step 10).
    Figure Legend Snippet: Model of phospholipid scramblase activation during HSV entry. Binding of HSV-1 glycoprotein C or HSV-2 glycoprotein B (Step 1) and engagement of nectin-1 by glycoprotein D (Step 2) trigger the release of a small amount of calcium near the plasma membrane (Step 3) that is sufficient to activate (phosphorylate) phospholipid scramblase (Step 4). This results in flipping of phosphatidylserines (PtdS) from the inner to the outer leaflet of the plasma membrane, which results in externalization of Akt and possibly other inner plasma membrane- associated proteins (Step 5). Akt interacts with gB, which may promote a conformational change in Akt that facilitates its phosphorylation at threonine 308 and serine 473 by yet to be determined kinases (Step 6). Integrinαvβ3 binds to gH (Step 7), and activated Akt and integrin transfer signals to the cell cytoplasm that lead to the generation of inositol triphosphates and release of inositol-triphosphate receptor (IP3R)-regulated Ca 2+ stores (Step 8) culminating in fusion of the viral envelope and plasma membrane and entry of viral capsids (Step 9). PtdS flip back to the inner leaflet of the plasma membrane to restore normal membrane asymmetry in a process that may occur in response to viral entry and may be regulated by gL binding to PLSCR1 and subsequent shift to primarily dephosphorylated PLSCR1. (Step 10).

    Techniques Used: Activation Assay, Binding Assay

    Viral binding to heparan sulfate and gD-nectin engagement precede and are required for activation of phospholipid scramblase. (A). CaSki or HaCATcells were transfected with siControl or siPLSCR1 and 72 h later were exposed to indicated multiplicities of infection (MOI) of HSV-2(G) for 4 hours at 4°C. The cells were then washed extensively and Western blots of cell lysates were probed with a mAb to gD as a marker of cell-bound virus, mAb for α-tubulin as a control for cell loading and rabbit anti-PLSCR1 as a probe for silencing. The blot is representative of results obtained in 2 independent experiments. (B). CaSki or HaCAT cells were transfected with control (Ctrl) or PLSCR1 siRNA and then infected with envelope-labeled (red) HSV-1VP26GFP (MOI 5 pfu/cell) for 4 hours at 4°C to detect binding or incubated for an additional 4 h at 37°C to detect viral entry. Results are representative of 2 independent experiments; bar = 10μm. (C). HSV-2(G) (~MOI 5 pfu/cell) was mixed with 100 μg/ml heparin or with 1:100 dilution of mAbs to HSV-2 glycoproteins gB, gD, gC, gL, or gH or control mouse IgG and then applied to CaSki cells that had been prestained with Alexa Fluor 594-conjugated wheat germ agglutinin to detect plasma membranes (red). After a 30-minute incubation, the cells were then washed, fixed and stained without permeabilization. Nuclei were stained blue with Hoechst and phosphatidylserines green with a primary murine and secondary Alexa 488-conjugated secondary antibody. Mock-infected cells are included as a control. Images are representative of results obtained from 3 independent experiments. (D). CaSki cells or HSV-2(G) (calculated to yield ~100 pfu/well) were pretreated (pre-rx) with antibodies to phosphatidylserine (PtdS), gD, Akt, nectin or control mouse IgG for 1 h and then washed 3 times (cells) or diluted 1:100 (virus-antibody mixture). Alternatively, the antibodies were added to cells at the time of viral infection for 1 h (entry). Cells were washed after the 1 h entry period, overlaid with fresh medium and plaques were quantified at 48 hours. Results are presented as pfu/well and are means+ SEM from duplicate wells in 2–3 individual experiments. The asterisks indicate p
    Figure Legend Snippet: Viral binding to heparan sulfate and gD-nectin engagement precede and are required for activation of phospholipid scramblase. (A). CaSki or HaCATcells were transfected with siControl or siPLSCR1 and 72 h later were exposed to indicated multiplicities of infection (MOI) of HSV-2(G) for 4 hours at 4°C. The cells were then washed extensively and Western blots of cell lysates were probed with a mAb to gD as a marker of cell-bound virus, mAb for α-tubulin as a control for cell loading and rabbit anti-PLSCR1 as a probe for silencing. The blot is representative of results obtained in 2 independent experiments. (B). CaSki or HaCAT cells were transfected with control (Ctrl) or PLSCR1 siRNA and then infected with envelope-labeled (red) HSV-1VP26GFP (MOI 5 pfu/cell) for 4 hours at 4°C to detect binding or incubated for an additional 4 h at 37°C to detect viral entry. Results are representative of 2 independent experiments; bar = 10μm. (C). HSV-2(G) (~MOI 5 pfu/cell) was mixed with 100 μg/ml heparin or with 1:100 dilution of mAbs to HSV-2 glycoproteins gB, gD, gC, gL, or gH or control mouse IgG and then applied to CaSki cells that had been prestained with Alexa Fluor 594-conjugated wheat germ agglutinin to detect plasma membranes (red). After a 30-minute incubation, the cells were then washed, fixed and stained without permeabilization. Nuclei were stained blue with Hoechst and phosphatidylserines green with a primary murine and secondary Alexa 488-conjugated secondary antibody. Mock-infected cells are included as a control. Images are representative of results obtained from 3 independent experiments. (D). CaSki cells or HSV-2(G) (calculated to yield ~100 pfu/well) were pretreated (pre-rx) with antibodies to phosphatidylserine (PtdS), gD, Akt, nectin or control mouse IgG for 1 h and then washed 3 times (cells) or diluted 1:100 (virus-antibody mixture). Alternatively, the antibodies were added to cells at the time of viral infection for 1 h (entry). Cells were washed after the 1 h entry period, overlaid with fresh medium and plaques were quantified at 48 hours. Results are presented as pfu/well and are means+ SEM from duplicate wells in 2–3 individual experiments. The asterisks indicate p

    Techniques Used: Binding Assay, Activation Assay, Transfection, Infection, Western Blot, Marker, Labeling, Incubation, Staining

    Phospholipid scramblase activation requires intracellular calcium and HSV-2 glycoprotein B and D. (A) CaSki cells were treated with 100μM BAPTA or BAPTA-AM and then infected with HSV-2(G) (10 pfu/cell) or mock-infected for 30 minutes. Cells were then fixed and stained with mAb to phosphatidylserines (PtdS) and secondary anti-mouse Alexa-555 (red), rabbit anti-PLSCR1 with Alexa-488 secondary Ab (green), or rabbit anti-Akt with Alexa-488 secondary Ab (green). Nuclei were stained blue with DAPI. Representative extended focus images from 2 independent experiments are shown (bar = 10μm). (B) CaSki cells were exposed to relatively equivalent particle numbers of HSV-2(G) or the indicated non-complemented deletion viruses (particle numbers estimated by comparing VP5 band on Western blots) for 30 minutes and fixed and stained as in panel A. Results are presentative of 4 independent experiments (2 experiments with ΔgL-2 -/- ). (C) The percentage of cells in which PtdS, Akt or PLSCR was detected by immunostaining was calculated by counting 80–100 cells in total from 4 random fields in at least 2 independent experiments (mean + SEM) and the asterisks indicate significant differences relative to HSV-2(G) (***p
    Figure Legend Snippet: Phospholipid scramblase activation requires intracellular calcium and HSV-2 glycoprotein B and D. (A) CaSki cells were treated with 100μM BAPTA or BAPTA-AM and then infected with HSV-2(G) (10 pfu/cell) or mock-infected for 30 minutes. Cells were then fixed and stained with mAb to phosphatidylserines (PtdS) and secondary anti-mouse Alexa-555 (red), rabbit anti-PLSCR1 with Alexa-488 secondary Ab (green), or rabbit anti-Akt with Alexa-488 secondary Ab (green). Nuclei were stained blue with DAPI. Representative extended focus images from 2 independent experiments are shown (bar = 10μm). (B) CaSki cells were exposed to relatively equivalent particle numbers of HSV-2(G) or the indicated non-complemented deletion viruses (particle numbers estimated by comparing VP5 band on Western blots) for 30 minutes and fixed and stained as in panel A. Results are presentative of 4 independent experiments (2 experiments with ΔgL-2 -/- ). (C) The percentage of cells in which PtdS, Akt or PLSCR was detected by immunostaining was calculated by counting 80–100 cells in total from 4 random fields in at least 2 independent experiments (mean + SEM) and the asterisks indicate significant differences relative to HSV-2(G) (***p

    Techniques Used: Activation Assay, Infection, Staining, Western Blot, Immunostaining

    8) Product Images from "Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition"

    Article Title: Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.13719

    Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.
    Figure Legend Snippet: Biotin‐Streptavidin affinity pulldown assay of RCZ12/20 with whole cell lysate of EHEC. A. Coomassie stained SDS‐PAGE gel of biotin‐RCZ12/20 bound proteins. Each wash and elution stage is indicated above each well. The negative control for nonspecific binding corresponds to the assay performed using Streptavidin beads alone. The experiment was performed in triplicate. B. The chemical structure of the biotin labeled RCZ12 and RCZ20 compounds used in the pull‐down assays. C. Table of results highlighting the targets of RCZ12/20 as identified by tandem mass spectrometry. The band number, genBank/protein ID, MOWSE score and gene name are indicated.

    Techniques Used: Staining, SDS Page, Negative Control, Binding Assay, Labeling, Mass Spectrometry

    Characterization of the affects of RCZ20 treatment on transcriptional regulation of type 3 secretion in EHEC. A. RNA‐seq results of EHEC cultured in MEM‐HEPES with and without RCZ20. A gene was determined as differentially expressed if it displayed significant upregulation or downregulation with an FDR corrected p value of
    Figure Legend Snippet: Characterization of the affects of RCZ20 treatment on transcriptional regulation of type 3 secretion in EHEC. A. RNA‐seq results of EHEC cultured in MEM‐HEPES with and without RCZ20. A gene was determined as differentially expressed if it displayed significant upregulation or downregulation with an FDR corrected p value of

    Techniques Used: RNA Sequencing Assay, Cell Culture

    9) Product Images from "Glucocorticoid and Growth Factor Synergism Requirement for Notch4 Chromatin Domain Activation ▿"

    Article Title: Glucocorticoid and Growth Factor Synergism Requirement for Notch4 Chromatin Domain Activation ▿

    Journal:

    doi: 10.1128/MCB.02152-06

    GR and AP-1 independently occupy a composite response element, consisting of an imperfect half-GRE and a neighboring AP-1 motif. (A) Sequences of 5′-biotinylated double-strand oligonucleotides used in the in vitro promoter complex assembly assay:
    Figure Legend Snippet: GR and AP-1 independently occupy a composite response element, consisting of an imperfect half-GRE and a neighboring AP-1 motif. (A) Sequences of 5′-biotinylated double-strand oligonucleotides used in the in vitro promoter complex assembly assay:

    Techniques Used: In Vitro

    10) Product Images from "AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination"

    Article Title: AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

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

    doi: 10.1073/pnas.0911879106

    CPT blocks CSR in CH12F3–2 cells and splenic B cells. ( A ) The percentages of the IgA + and propidium iodide (PI) − alive cells are presented as closed and open circles, respectively. Each circle represents average with S.D. ( n = 3). The backgrounds (subtracted) of IgA + and PI + cells at 0 nM CPT were 1.26 and 11.5%, respectively. ( B ) AID −/− spleen cells were infected by AIDER-IRES-GFP retrovirus. Twenty-four hours later, the cells were treated with 1 μM 4-OHT or EtOH in the presence of 30 to 60 nM CPT. After an additional 24-h incubation, surface IgG1 and GFP expression were analyzed by flow cytometry with biotinylated anti-IgG1 antibody and streptavidin-APC. The percentage of IgG1 switch in the GFP positive cells is shown above each plot. Mock, no retrovirus control. ( C ) S γ 1-S μ DC-PCR. Before the flow cytometry in panel B , part of cells were collected 3 and 6 h after 4-OHT and CPT addition. Extracted genomic DNA was then subjected to DC-PCR for the S γ 1-S μ recombination (γ 1 -μ) and for the nicotinic acetylcholine receptor ( nAchR ) locus as control. PCR products were run on a 2% agarose gel and visualized by ethidium bromide.
    Figure Legend Snippet: CPT blocks CSR in CH12F3–2 cells and splenic B cells. ( A ) The percentages of the IgA + and propidium iodide (PI) − alive cells are presented as closed and open circles, respectively. Each circle represents average with S.D. ( n = 3). The backgrounds (subtracted) of IgA + and PI + cells at 0 nM CPT were 1.26 and 11.5%, respectively. ( B ) AID −/− spleen cells were infected by AIDER-IRES-GFP retrovirus. Twenty-four hours later, the cells were treated with 1 μM 4-OHT or EtOH in the presence of 30 to 60 nM CPT. After an additional 24-h incubation, surface IgG1 and GFP expression were analyzed by flow cytometry with biotinylated anti-IgG1 antibody and streptavidin-APC. The percentage of IgG1 switch in the GFP positive cells is shown above each plot. Mock, no retrovirus control. ( C ) S γ 1-S μ DC-PCR. Before the flow cytometry in panel B , part of cells were collected 3 and 6 h after 4-OHT and CPT addition. Extracted genomic DNA was then subjected to DC-PCR for the S γ 1-S μ recombination (γ 1 -μ) and for the nicotinic acetylcholine receptor ( nAchR ) locus as control. PCR products were run on a 2% agarose gel and visualized by ethidium bromide.

    Techniques Used: Cycling Probe Technology, Infection, Incubation, Expressing, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    11) Product Images from "Structural and functional impacts of ER coactivator sequential recruitment"

    Article Title: Structural and functional impacts of ER coactivator sequential recruitment

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.07.026

    ER sequentially recruits different coactivators. (a) CARM1 recruitment to the ER complex followed behind the recruitment of SRC-3. Shown are the ChIP results of ERα, SRC-3 and CARM1 in MCF-7 cells stimulated with 10nM E2 at different time points. (b) CARM1 synergized with SRC-3 and p300 to activate ER-mediated transcription. The ER-mediated transcription activity was measured through an ERE-driven luciferase reporter in the absence or presence of 10nM 17-β estradiol (E2). Data are represented as mean ± SEM.
    Figure Legend Snippet: ER sequentially recruits different coactivators. (a) CARM1 recruitment to the ER complex followed behind the recruitment of SRC-3. Shown are the ChIP results of ERα, SRC-3 and CARM1 in MCF-7 cells stimulated with 10nM E2 at different time points. (b) CARM1 synergized with SRC-3 and p300 to activate ER-mediated transcription. The ER-mediated transcription activity was measured through an ERE-driven luciferase reporter in the absence or presence of 10nM 17-β estradiol (E2). Data are represented as mean ± SEM.

    Techniques Used: Chromatin Immunoprecipitation, Activity Assay, Luciferase

    Workflow for stepwise 3D classification of ERα + SRC-3 + p300 + CARM1 (a) and of ERα + SRC-3 + p300 + CARM1 + Fab (b) datasets using new multirefine and e2refinemulti in EMAN2. Each subset was refined separately with specified particle number. Each map was segmented to annotate different proteins with p300 in Blue, ER in Green, SRC-3a/b in Orange and Red, respectively, CARM1 in Pink and CARM1 Fab in Yellow.
    Figure Legend Snippet: Workflow for stepwise 3D classification of ERα + SRC-3 + p300 + CARM1 (a) and of ERα + SRC-3 + p300 + CARM1 + Fab (b) datasets using new multirefine and e2refinemulti in EMAN2. Each subset was refined separately with specified particle number. Each map was segmented to annotate different proteins with p300 in Blue, ER in Green, SRC-3a/b in Orange and Red, respectively, CARM1 in Pink and CARM1 Fab in Yellow.

    Techniques Used:

    CARM1 overexpression reduced SRC-3:ER ratio and its N-terminal domain mediated the interaction with p300. (a) Raw Flow Proteometric analysis data for single ER/SRC-3 complex in HeLa cells without (left panel) or with (right panel) CARM1 overexpression. (b) Box Whisker Plot of SRC-3 and ERα fluorescence intensities detected in single ER/SRC-3 complexes without or with CARM1 expression. (c) The N-terminal domain of CARM1 (1-140aa) mediated the interaction between CARM1 and p300 in vitro. Shown is the GST pull-down experiment using GST-fused CARM1 full length and different fragments to pull down purified p300 protein. (d) CARM1 N-terminal domain deletion significantly reduced its ability to interact with p300 and ER in cells. Shown is a co-IP experiment.
    Figure Legend Snippet: CARM1 overexpression reduced SRC-3:ER ratio and its N-terminal domain mediated the interaction with p300. (a) Raw Flow Proteometric analysis data for single ER/SRC-3 complex in HeLa cells without (left panel) or with (right panel) CARM1 overexpression. (b) Box Whisker Plot of SRC-3 and ERα fluorescence intensities detected in single ER/SRC-3 complexes without or with CARM1 expression. (c) The N-terminal domain of CARM1 (1-140aa) mediated the interaction between CARM1 and p300 in vitro. Shown is the GST pull-down experiment using GST-fused CARM1 full length and different fragments to pull down purified p300 protein. (d) CARM1 N-terminal domain deletion significantly reduced its ability to interact with p300 and ER in cells. Shown is a co-IP experiment.

    Techniques Used: Over Expression, Flow Cytometry, Whisker Assay, Fluorescence, Expressing, In Vitro, Purification, Co-Immunoprecipitation Assay

    Communication between p300 and CARM1 within the ER complex regulates HAT and HMT activity. (a) Segmented p300 from map 1A. (b) Segmented p300 from map 1C. (c) CARM1 increased p300 autoacetylation and its HAT activity on histone H3 in vitro. Shown is the autoradiograph of 3 H-labeled acetylation. (d) Coomassie blue staining of purified GST-CARM1 WT and Δ1-140 mutant from bacteria. (e) Deletion of the N-terminal domain abolished the ability of CARM1 to regulate p300 HAT activity in vitro. Top panel, the autoradiograph of 3 H-labeled acetylation. Bottom panels, Western blot analysis on the levels of ER, SRC-3, p300 and CARM1 WT or Δ1-140 mutant in the input. * represents the uncleaved GST-CARM1. # represents GST-removed CARM1 through thrombin cleavage. (f) CARM1 selectively increased p300 HAT activity on H3K18. Shown is the Western blot analysis of the levels of histone acetylation in the in vitro HAT assay using different acetylation antibodies. (g) p300-mediated acetylation significantly increased CARM1-mediated H3R17 methylation. Shown is the autoradiograph of 3 H-labeled methylation in vitro.
    Figure Legend Snippet: Communication between p300 and CARM1 within the ER complex regulates HAT and HMT activity. (a) Segmented p300 from map 1A. (b) Segmented p300 from map 1C. (c) CARM1 increased p300 autoacetylation and its HAT activity on histone H3 in vitro. Shown is the autoradiograph of 3 H-labeled acetylation. (d) Coomassie blue staining of purified GST-CARM1 WT and Δ1-140 mutant from bacteria. (e) Deletion of the N-terminal domain abolished the ability of CARM1 to regulate p300 HAT activity in vitro. Top panel, the autoradiograph of 3 H-labeled acetylation. Bottom panels, Western blot analysis on the levels of ER, SRC-3, p300 and CARM1 WT or Δ1-140 mutant in the input. * represents the uncleaved GST-CARM1. # represents GST-removed CARM1 through thrombin cleavage. (f) CARM1 selectively increased p300 HAT activity on H3K18. Shown is the Western blot analysis of the levels of histone acetylation in the in vitro HAT assay using different acetylation antibodies. (g) p300-mediated acetylation significantly increased CARM1-mediated H3R17 methylation. Shown is the autoradiograph of 3 H-labeled methylation in vitro.

    Techniques Used: HAT Assay, HMT Assay, Activity Assay, In Vitro, Autoradiography, Labeling, Staining, Purification, Mutagenesis, Western Blot, Methylation

    12) Product Images from "Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli"

    Article Title: Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149718

    A schematic model of the Pch-mediated activation of the LEE1 promoter. The nucleoid complex is composed of H-NS, StpA, Hha and YdgT. The role of YdgT is uncertain but it could be a member of the complex. In the silencing complex, DNA is folded through bridging between the proteins, and RNA polymerase might be trapped (silent). Competitive binding of Pch removes some H-NS and other proteins from the complex or inhibits the binding of them, resulting in the relaxed complex form, in which RNA polymerase can start transcription (active).
    Figure Legend Snippet: A schematic model of the Pch-mediated activation of the LEE1 promoter. The nucleoid complex is composed of H-NS, StpA, Hha and YdgT. The role of YdgT is uncertain but it could be a member of the complex. In the silencing complex, DNA is folded through bridging between the proteins, and RNA polymerase might be trapped (silent). Competitive binding of Pch removes some H-NS and other proteins from the complex or inhibits the binding of them, resulting in the relaxed complex form, in which RNA polymerase can start transcription (active).

    Techniques Used: Activation Assay, Binding Assay

    Changes in H-NS-containing nucleoprotein complexes by pchA expression. A. Difference in the effect of PchA on H-NS binding at the LEE1 promoter region. ChIP-purified DNA from EHEC expressing PchA (Pch+) or deficient in pch (Pch-) was used as templates in PCR for various segments (1 to 5). B. Difference in sensitivity to hydroxyl radical attack. ChIP-purified H-NS-DNA complexes were incubated with hydroxyl radicals for 0–4 min, and then the DNA was purified. Two segments (1 and 2) of the LEE1 promoter region were detected by semi-quantitative PCR. As a control, from the same samples, DNA segment of gadE promoter region (P gadE ) were detected by PCR. C. Effect of H-NS/StpA on the binding of PchA to the LEE1 promoter region. ChIP-purified PchA-bound DNA from the W3110 wild type or the hns stpA mutant harboring pLux-P LEE1 and pTB101- pchA -Strep were used as PCR templates for various segments (1 to 4).
    Figure Legend Snippet: Changes in H-NS-containing nucleoprotein complexes by pchA expression. A. Difference in the effect of PchA on H-NS binding at the LEE1 promoter region. ChIP-purified DNA from EHEC expressing PchA (Pch+) or deficient in pch (Pch-) was used as templates in PCR for various segments (1 to 5). B. Difference in sensitivity to hydroxyl radical attack. ChIP-purified H-NS-DNA complexes were incubated with hydroxyl radicals for 0–4 min, and then the DNA was purified. Two segments (1 and 2) of the LEE1 promoter region were detected by semi-quantitative PCR. As a control, from the same samples, DNA segment of gadE promoter region (P gadE ) were detected by PCR. C. Effect of H-NS/StpA on the binding of PchA to the LEE1 promoter region. ChIP-purified PchA-bound DNA from the W3110 wild type or the hns stpA mutant harboring pLux-P LEE1 and pTB101- pchA -Strep were used as PCR templates for various segments (1 to 4).

    Techniques Used: Expressing, Binding Assay, Chromatin Immunoprecipitation, Purification, Polymerase Chain Reaction, Incubation, Real-time Polymerase Chain Reaction, Mutagenesis

    Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.
    Figure Legend Snippet: Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Techniques Used: Incubation, Magnetic Beads, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    13) Product Images from "On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis"

    Article Title: On-chip magnetic separation of superparamagnetic beads for integrated molecular analysis

    Journal: Journal of Applied Physics

    doi: 10.1063/1.3272779

    Magnetic bead binding chemistry. Surface polyclonal goat IgG specific to the F ab region of human IgG is passively adsorbed on the gold surface. Human IgG antigen is added, followed by the primary biotinylated monoclonal goat IgG specific to the F c region of the human IgG antigen. Last, the streptavidin-coated 4.5 μm Dynal bead labels are added.
    Figure Legend Snippet: Magnetic bead binding chemistry. Surface polyclonal goat IgG specific to the F ab region of human IgG is passively adsorbed on the gold surface. Human IgG antigen is added, followed by the primary biotinylated monoclonal goat IgG specific to the F c region of the human IgG antigen. Last, the streptavidin-coated 4.5 μm Dynal bead labels are added.

    Techniques Used: Binding Assay

    14) Product Images from "Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection"

    Article Title: Specific Magnetic Isolation of E6 HPV16 Modified Magnetizable Particles Coupled with PCR and Electrochemical Detection

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17050585

    Scheme of DNA nanoconstruct of biotin-modified oligonucleotides bound to E6-HPV16 oncogene joined to streptavidin modified MPs : ( A ) 100 µL (10 mg·mL −1 ) of commercial Dynabeads M-280 Streptavidin MPs; ( B ) E6-HPV16 complementary oligonucleotides biotinylated (forward and reverse) (20 µL, 100 µM) using Biotin 3′ end DNA Labeling Kit (Thermo Scientific, Waltham, MA, USA) were successfully conjugated with Dynabeads; and ( C ) E6-HPV16 DNA was amplified from E6-HPV16-pUC57 synthetic plasmid by PCR, which was subsequently purified and conjugated with the nanoconstruct.
    Figure Legend Snippet: Scheme of DNA nanoconstruct of biotin-modified oligonucleotides bound to E6-HPV16 oncogene joined to streptavidin modified MPs : ( A ) 100 µL (10 mg·mL −1 ) of commercial Dynabeads M-280 Streptavidin MPs; ( B ) E6-HPV16 complementary oligonucleotides biotinylated (forward and reverse) (20 µL, 100 µM) using Biotin 3′ end DNA Labeling Kit (Thermo Scientific, Waltham, MA, USA) were successfully conjugated with Dynabeads; and ( C ) E6-HPV16 DNA was amplified from E6-HPV16-pUC57 synthetic plasmid by PCR, which was subsequently purified and conjugated with the nanoconstruct.

    Techniques Used: Modification, DNA Labeling, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Purification

    15) Product Images from "rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences"

    Article Title: rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04370-x

    rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing

    Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f
    Figure Legend Snippet: Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f

    Techniques Used: Binding Assay, Incubation, Labeling, In Vitro, Silver Staining, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

    rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, CTL Assay, Reverse Transcription Polymerase Chain Reaction, FACS

    16) Product Images from "Bortezomib-inducible long non-coding RNA myocardial infarction associated transcript is an oncogene in multiple myeloma that suppresses miR-29b"

    Article Title: Bortezomib-inducible long non-coding RNA myocardial infarction associated transcript is an oncogene in multiple myeloma that suppresses miR-29b

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-1551-z

    Knockdown of MIAT sensitizes MM cells to BTZ. a CCK-8 assays were used to determine cell viability after sh-MIAT lentivirus infection. b Flow cytometry was used to determine apoptosis after knockdown of MIAT. c Western blot analyses of apoptosis markers Bcl-2, Bak1, and cleaved PARP after knockdown of MIAT. d Changes of in half inhibitory concentrations (IC 50 ) of BTZ in U266 cells after knockdown of MIAT were determined using CCK-8 assays. e Changes in IC 50 of BTZ in KM3 cells after knockdown of MIAT. f U266 cells were infected with MIAT shRNA lentivirus and were then injected into nude mice. Cells transfected with empty lentivirus were used as a negative control. Mice were euthanized and tumors were collected on day 28 after injection. Tumor volumes were measured every week. g Ki67 expression in tumor sections was evaluated using immunohistochemistry (IHC); Bar, 50 μm; * P
    Figure Legend Snippet: Knockdown of MIAT sensitizes MM cells to BTZ. a CCK-8 assays were used to determine cell viability after sh-MIAT lentivirus infection. b Flow cytometry was used to determine apoptosis after knockdown of MIAT. c Western blot analyses of apoptosis markers Bcl-2, Bak1, and cleaved PARP after knockdown of MIAT. d Changes of in half inhibitory concentrations (IC 50 ) of BTZ in U266 cells after knockdown of MIAT were determined using CCK-8 assays. e Changes in IC 50 of BTZ in KM3 cells after knockdown of MIAT. f U266 cells were infected with MIAT shRNA lentivirus and were then injected into nude mice. Cells transfected with empty lentivirus were used as a negative control. Mice were euthanized and tumors were collected on day 28 after injection. Tumor volumes were measured every week. g Ki67 expression in tumor sections was evaluated using immunohistochemistry (IHC); Bar, 50 μm; * P

    Techniques Used: CCK-8 Assay, Infection, Flow Cytometry, Cytometry, Western Blot, shRNA, Injection, Mouse Assay, Transfection, Negative Control, Expressing, Immunohistochemistry

    MIAT negatively regulates miR-29b. a Luciferase reporter gene assays of library miRNAs were performed to screen for MIAT targets. b – d Correlations between MIAT and miR-29b, miR-489, and miR-150 in patients with MM. e qRT-PCR analyses of miR-29b, miR-489, and miR-150 expression in U266 cells after infection with sh-MIAT lentivirus or empty control vector. f Sequence alignments of miR-29b with putative binding sites within wild type (WT; red) and mutant regions (blue) of MIAT. g Relative luciferase activities were inhibited in U266 cells that were co-transfected with the wild-type MIAT 3′UTR vector and a miR-29b mimic, but not with the mutant-type vector. Firefly luciferase activity was normalized to that of Renilla luciferase. h Levels of MIAT in samples that were pulled down using biotinylated miR-29b were measured using real-time PCR. i miR-29b expression levels in samples that were pulled down using biotinylated MIAT were measured using real-time PCR. j U266 cells were transfected with miR-29b mimic, or with wild type (WT) or mutant (MUT) MIAT for 48 h, and miR-29b expression was determined using real-time PCR. k Real-time PCR analysis of validated miR-29b targets after indicated treatments. l Western blotting analyses of validated miR-29b targets after indicated treatments; GAPDH was used a loading control; * P
    Figure Legend Snippet: MIAT negatively regulates miR-29b. a Luciferase reporter gene assays of library miRNAs were performed to screen for MIAT targets. b – d Correlations between MIAT and miR-29b, miR-489, and miR-150 in patients with MM. e qRT-PCR analyses of miR-29b, miR-489, and miR-150 expression in U266 cells after infection with sh-MIAT lentivirus or empty control vector. f Sequence alignments of miR-29b with putative binding sites within wild type (WT; red) and mutant regions (blue) of MIAT. g Relative luciferase activities were inhibited in U266 cells that were co-transfected with the wild-type MIAT 3′UTR vector and a miR-29b mimic, but not with the mutant-type vector. Firefly luciferase activity was normalized to that of Renilla luciferase. h Levels of MIAT in samples that were pulled down using biotinylated miR-29b were measured using real-time PCR. i miR-29b expression levels in samples that were pulled down using biotinylated MIAT were measured using real-time PCR. j U266 cells were transfected with miR-29b mimic, or with wild type (WT) or mutant (MUT) MIAT for 48 h, and miR-29b expression was determined using real-time PCR. k Real-time PCR analysis of validated miR-29b targets after indicated treatments. l Western blotting analyses of validated miR-29b targets after indicated treatments; GAPDH was used a loading control; * P

    Techniques Used: Luciferase, Quantitative RT-PCR, Expressing, Infection, Plasmid Preparation, Sequencing, Binding Assay, Mutagenesis, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

    miR-29b inhibitor reverses the inhibitory effects of MIAT downregulation in U266 cells. a Apoptosis was evaluated using flow cytometry after treating U266 cells with miR-29b inhibitor and MIAT shRNA. b Changes in IC 50 values for BTZ in U266 cells were determined using CCK-8 assays after treatments with miR-29b inhibitor and MIAT shRNA; * P
    Figure Legend Snippet: miR-29b inhibitor reverses the inhibitory effects of MIAT downregulation in U266 cells. a Apoptosis was evaluated using flow cytometry after treating U266 cells with miR-29b inhibitor and MIAT shRNA. b Changes in IC 50 values for BTZ in U266 cells were determined using CCK-8 assays after treatments with miR-29b inhibitor and MIAT shRNA; * P

    Techniques Used: Flow Cytometry, Cytometry, shRNA, CCK-8 Assay

    BTZ upregulates MIAT in MM cells via p38-Stat1 signaling. a–e U266 cells were pretreated with the selective pharmacological inhibitors U0126 (ERK, 50 μM), SP600125 (JNK, 50 μM), Bay-11-7082 (NF-κB, 10 μM), SB203580 (p38, 50 μM), or PF-04965842 (Jak1, 50 nM), and were then treated with BTZ at 40 nM for 12 h. MIAT expression levels were determined using qRT-PCR. f Western blotting analyses of Stat1 and phosphorylated Stat1 after overexpression or knockdown in U266 cells. g Effects of Stat1 overexpression on MIAT expression in U266 cells. h Effects of Stat1 knockdown on MIAT expression in U266 cells. i Effects of p38 overexpression on MIAT expression in U266 cells pretreated with sh-NC or sh-Stat1. j Luciferase reporter constructs containing the MIAT promoter were co-transfected into U266 cells with the internal control plasmid pRL-TK, and with sh-NC or sh-Stat1, and were then subjected to BTZ challenge (40 nM, 12 h). Relative luciferase activities are expressed as percentages of those in the control group. k Cell lysates from U266 cells were used for RIP with antibodies against stat1, stat3, or NF-κB. MIAT expression levels were detected using qRT-PCR. IgG was used as a negative control. l BTZ induced Stat1 phosphorylation; data are presented as means ± standard errors of the mean from three independent experiments; * P
    Figure Legend Snippet: BTZ upregulates MIAT in MM cells via p38-Stat1 signaling. a–e U266 cells were pretreated with the selective pharmacological inhibitors U0126 (ERK, 50 μM), SP600125 (JNK, 50 μM), Bay-11-7082 (NF-κB, 10 μM), SB203580 (p38, 50 μM), or PF-04965842 (Jak1, 50 nM), and were then treated with BTZ at 40 nM for 12 h. MIAT expression levels were determined using qRT-PCR. f Western blotting analyses of Stat1 and phosphorylated Stat1 after overexpression or knockdown in U266 cells. g Effects of Stat1 overexpression on MIAT expression in U266 cells. h Effects of Stat1 knockdown on MIAT expression in U266 cells. i Effects of p38 overexpression on MIAT expression in U266 cells pretreated with sh-NC or sh-Stat1. j Luciferase reporter constructs containing the MIAT promoter were co-transfected into U266 cells with the internal control plasmid pRL-TK, and with sh-NC or sh-Stat1, and were then subjected to BTZ challenge (40 nM, 12 h). Relative luciferase activities are expressed as percentages of those in the control group. k Cell lysates from U266 cells were used for RIP with antibodies against stat1, stat3, or NF-κB. MIAT expression levels were detected using qRT-PCR. IgG was used as a negative control. l BTZ induced Stat1 phosphorylation; data are presented as means ± standard errors of the mean from three independent experiments; * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Luciferase, Construct, Transfection, Plasmid Preparation, Negative Control

    17) Product Images from "Local palmitoylation cycles define activity-regulated postsynaptic subdomains"

    Article Title: Local palmitoylation cycles define activity-regulated postsynaptic subdomains

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201302071

    DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.
    Figure Legend Snippet: DHHC2 directly nucleates PSD-95 assembly at the plasma membrane through local palmitoylation. (A) HEK293T cells were cotransfected with a bi-cistronic RUSH vector containing streptavidin-Ii (Str-ER Hook) and streptavidin-binding peptide (SBP)-GFP-DHHC2 as well as PSD-95-mCherry. Synchronized release of DHHC2 from the ER was induced by the addition of biotin with or without 2-BP. Arrowheads denote signals at the plasma membrane. Bar, 10 µm. (B) Kymograph analysis. The fluorescence intensities of GFP and mCherry were measured along red lines in A. White arrows indicate the timing when DHHC2 arrived at the plasma membrane. Black arrows indicate the position of the plasma membrane (at 90 min). CS, inactive DHHC2. Bar, 2.5 µm.

    Techniques Used: Plasmid Preparation, Binding Assay, Fluorescence

    18) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    19) Product Images from "RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element"

    Article Title: RNA-Binding Motif Protein 24 (RBM24) Is Involved in Pregenomic RNA Packaging by Mediating Interaction between Hepatitis B Virus Polymerase and the Epsilon Element

    Journal: Journal of Virology

    doi: 10.1128/JVI.02161-18

    RBM24 specifically binds to HBV ε. HEK293T cells were transfected with 24 μg of pRBM24 in 100-mm dishes, and cell lysates containing RBM24 protein were harvested at 48 hpt. Recombinant human RBM24 (rhRBM24) proteins were purified in vitro . (A) Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled p21-ARE (positive control), yeast tRNA (negative control), the TR, or the TRΔε fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. IB, immunoblotting. (B) Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled p21-ARE, yeast tRNA, or the ε RNA fragment. An excess amount of cold unlabeled ε RNA (0×, 10×, or 20×) was added to compete with the binding of RBM24 to the biotin-labeled ε fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. (C) The apical loop and internal bulge structural elements were deleted from full-length ε [FL(ε)] to generate FLΔA and FLΔB, respectively. Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled ε, FLΔA, or FLΔB, followed by pulldown with streptavidin beads and assessment by Western blotting. (D) The mutation bulge structural elements, B1-6, ApaB, B1-4, B35, B2346, B15, B1A, B1U, B1G, B5A, B5C, and B5G were constructed, Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled ε mutations. followed by pulldown with streptavidin beads and assessment by Western blotting.
    Figure Legend Snippet: RBM24 specifically binds to HBV ε. HEK293T cells were transfected with 24 μg of pRBM24 in 100-mm dishes, and cell lysates containing RBM24 protein were harvested at 48 hpt. Recombinant human RBM24 (rhRBM24) proteins were purified in vitro . (A) Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled p21-ARE (positive control), yeast tRNA (negative control), the TR, or the TRΔε fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. IB, immunoblotting. (B) Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled p21-ARE, yeast tRNA, or the ε RNA fragment. An excess amount of cold unlabeled ε RNA (0×, 10×, or 20×) was added to compete with the binding of RBM24 to the biotin-labeled ε fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. (C) The apical loop and internal bulge structural elements were deleted from full-length ε [FL(ε)] to generate FLΔA and FLΔB, respectively. Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled ε, FLΔA, or FLΔB, followed by pulldown with streptavidin beads and assessment by Western blotting. (D) The mutation bulge structural elements, B1-6, ApaB, B1-4, B35, B2346, B15, B1A, B1U, B1G, B5A, B5C, and B5G were constructed, Lysates (400 μg) of cells transfected with pRBM24 or 50 pM rhRBM24 were incubated with 2 μg of biotin-labeled ε mutations. followed by pulldown with streptavidin beads and assessment by Western blotting.

    Techniques Used: Transfection, Recombinant, Purification, In Vitro, Incubation, Labeling, Positive Control, Negative Control, Western Blot, Binding Assay, Mutagenesis, Construct

    20) Product Images from "Dynamic m6A mRNA methylation directs translational control of heat shock response"

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response

    Journal: Nature

    doi: 10.1038/nature15377

    YTHDF2 changes cellular localization and expression levels in response to heat shock stress a , Schematic of m 6 A modification machinery in mammalian cells. b , Subcellular localization of YTHDF2 in MEF and HeLa cells before or 2 h after heat shock (42°C, 1 h). Bar, 10 μm. Representative of at least 50 cells. c , Immunoblotting of MEF cells after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , Same samples in c were used for RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: YTHDF2 changes cellular localization and expression levels in response to heat shock stress a , Schematic of m 6 A modification machinery in mammalian cells. b , Subcellular localization of YTHDF2 in MEF and HeLa cells before or 2 h after heat shock (42°C, 1 h). Bar, 10 μm. Representative of at least 50 cells. c , Immunoblotting of MEF cells after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , Same samples in c were used for RNA extraction and real-time PCR. Relative levels of indicated transcripts are normalized to β-actin. Error bars, mean ± s.e.m.; * p

    Techniques Used: Expressing, Modification, RNA Extraction, Real-time Polymerase Chain Reaction

    m 6 A modification promotes selective translation under heat shock stress a, A 3-D plot depicting fold changes (log 2 ) of mRNA abundance, CDS ribosome occupancy, and 5′UTR m 6 A levels in MEF cells after heat shock stress. b , m 6 A blotting of HSPA1A purified from MEF with or without YTHDF2 knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. Representative of two biological replicates. c , Immunoblotting of MEF cells with or without YTHDF2 knockdown after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , MEF cells with or without YTHDF2 knockdown were subject to heat shock stress followed by sucrose gradient sedimentation. Specific mRNA levels in polysome fractions were measured by qPCR. The values are first normalized to the spike in control then to the total. Error bars, mean ± s.e.m.; * p
    Figure Legend Snippet: m 6 A modification promotes selective translation under heat shock stress a, A 3-D plot depicting fold changes (log 2 ) of mRNA abundance, CDS ribosome occupancy, and 5′UTR m 6 A levels in MEF cells after heat shock stress. b , m 6 A blotting of HSPA1A purified from MEF with or without YTHDF2 knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. Representative of two biological replicates. c , Immunoblotting of MEF cells with or without YTHDF2 knockdown after heat shock stress (42°C, 1 h). N: no heat shock. The right panel shows the relative protein levels quantified by densitometry and normalized to β-actin. Representative of three biological replicates. d , MEF cells with or without YTHDF2 knockdown were subject to heat shock stress followed by sucrose gradient sedimentation. Specific mRNA levels in polysome fractions were measured by qPCR. The values are first normalized to the spike in control then to the total. Error bars, mean ± s.e.m.; * p

    Techniques Used: Modification, Purification, Synthesized, In Vitro, Sedimentation, Real-time Polymerase Chain Reaction

    Direct competition between YTHDF2 and FTO in m 6 A binding a, Synthesized mRNA with m 6 A was incubated with FTO (2 μg) in the presence of increasing amount of YTHDF2 (0, 0.5, 1, 2 μg), followed by RNA pulldown and immunoblotting. b , Synthesized mRNA with m 6 A was incubated with FTO (1 μg in top panel and 2 μg in bottom panel) in the absence of presence of YTHDF2 (4 μg), followed by m 6 A dot blotting.
    Figure Legend Snippet: Direct competition between YTHDF2 and FTO in m 6 A binding a, Synthesized mRNA with m 6 A was incubated with FTO (2 μg) in the presence of increasing amount of YTHDF2 (0, 0.5, 1, 2 μg), followed by RNA pulldown and immunoblotting. b , Synthesized mRNA with m 6 A was incubated with FTO (1 μg in top panel and 2 μg in bottom panel) in the absence of presence of YTHDF2 (4 μg), followed by m 6 A dot blotting.

    Techniques Used: Binding Assay, Synthesized, Incubation

    FTO knockdown promotes Hsp70 synthesis a, m 6 A blotting of purified HSPA1A in MEF with or without FTO knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. RNA staining is shown as loading control. Representative of two biological replicates. b , MEF cells with or without FTO knockdown were collected at indicated times after heat shock stress (42°C, 1 h) followed by immunoblotting using antibodies indicated. N: no heat shock. Representative of three biological replicates.
    Figure Legend Snippet: FTO knockdown promotes Hsp70 synthesis a, m 6 A blotting of purified HSPA1A in MEF with or without FTO knockdown. mRNAs synthesized by in vitro transcription in the absence or presence of m 6 A were used as control. RNA staining is shown as loading control. Representative of two biological replicates. b , MEF cells with or without FTO knockdown were collected at indicated times after heat shock stress (42°C, 1 h) followed by immunoblotting using antibodies indicated. N: no heat shock. Representative of three biological replicates.

    Techniques Used: Purification, Synthesized, In Vitro, Staining

    21) Product Images from "A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification"

    Article Title: A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115046

    The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.
    Figure Legend Snippet: The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.

    Techniques Used: Concentration Assay, Microscopy, Standard Deviation

    Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.
    Figure Legend Snippet: Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Techniques Used: Labeling, Biomarker Assay, Concentration Assay

    Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.
    Figure Legend Snippet: Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Techniques Used: Fluorescence, Microscopy, Labeling, Biomarker Assay, Concentration Assay

    22) Product Images from "Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins"

    Article Title: Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.21.10090-10105.2001

    The vaccinia virus I1 protein binds telomeres and is responsible for the upper doublet of shifted complexes. (A and B) Identification of I1 by affinity purification. The 200-bp viral hairpins were biotinylated and conjugated to streptavidin-coated magnetic beads. Hairpin beads were then incubated with an infected cell extract using conditions similar to those used in EMSA reactions, including the presence of poly(dI-dC) and 150 mM KCl. Beads were collected using a magnet and developed with buffer containing increasing concentrations of KCl. Washes were assayed for telomere binding activity by EMSA using the 65-bp+tet hairpin probe (A) and analyzed in parallel by SDS-PAGE and silver staining (B). Lanes 1, cytoplasmic extract before incubation with beads; lanes 2, cytoplasmic extract after incubation with beads (flow through); lanes 3 and 4, 150 mM KCl washes; lanes 5, 250 mM KCl wash; lanes 6, 500 mM KCl wash; lanes 7, 1,000 mM KCl wash. The 35-kDa band in the 500 mM KCl wash (panel B, lane 6, lower gray arrow) was excised and identified as the vaccinia virus I1 protein by mass spectroscopy (see the text). Protein standards are shown at the right with their molecular masses indicated in kilodaltons. (C) The vaccinia virus I1 protein is necessary and sufficient for complex formation. Cytoplasmic extracts from uninfected cells (lane 1) or infected cells harvested at 24 hpi (lane 2 to 4) were analyzed by EMSA using the 65-bp+tet hairpin probe (upper panel) and by immunoblot analysis using a polyclonal anti-I1 serum (lower panel). Cells were infected with the following: lane 2, wt virus (wtVV); lane 3, vLacI (a virus expressing the lac repressor protein); lane 4, v ind I1 in the absence of IPTG. In lane 5, 320 ng of His-tagged recombinant I1 protein (HisI1) was used in the EMSA reaction (upper panel) and immunoblot analysis (lower panel). Dots and black arrows in panels A and C indicate the upper and lower doublets of shifted complexes, respectively; gray arrows in panel B indicate the 35- and 40-kDa proteins discussed in the text. Protein standards are shown at the right with their molecular masses indicated in kilodaltons.
    Figure Legend Snippet: The vaccinia virus I1 protein binds telomeres and is responsible for the upper doublet of shifted complexes. (A and B) Identification of I1 by affinity purification. The 200-bp viral hairpins were biotinylated and conjugated to streptavidin-coated magnetic beads. Hairpin beads were then incubated with an infected cell extract using conditions similar to those used in EMSA reactions, including the presence of poly(dI-dC) and 150 mM KCl. Beads were collected using a magnet and developed with buffer containing increasing concentrations of KCl. Washes were assayed for telomere binding activity by EMSA using the 65-bp+tet hairpin probe (A) and analyzed in parallel by SDS-PAGE and silver staining (B). Lanes 1, cytoplasmic extract before incubation with beads; lanes 2, cytoplasmic extract after incubation with beads (flow through); lanes 3 and 4, 150 mM KCl washes; lanes 5, 250 mM KCl wash; lanes 6, 500 mM KCl wash; lanes 7, 1,000 mM KCl wash. The 35-kDa band in the 500 mM KCl wash (panel B, lane 6, lower gray arrow) was excised and identified as the vaccinia virus I1 protein by mass spectroscopy (see the text). Protein standards are shown at the right with their molecular masses indicated in kilodaltons. (C) The vaccinia virus I1 protein is necessary and sufficient for complex formation. Cytoplasmic extracts from uninfected cells (lane 1) or infected cells harvested at 24 hpi (lane 2 to 4) were analyzed by EMSA using the 65-bp+tet hairpin probe (upper panel) and by immunoblot analysis using a polyclonal anti-I1 serum (lower panel). Cells were infected with the following: lane 2, wt virus (wtVV); lane 3, vLacI (a virus expressing the lac repressor protein); lane 4, v ind I1 in the absence of IPTG. In lane 5, 320 ng of His-tagged recombinant I1 protein (HisI1) was used in the EMSA reaction (upper panel) and immunoblot analysis (lower panel). Dots and black arrows in panels A and C indicate the upper and lower doublets of shifted complexes, respectively; gray arrows in panel B indicate the 35- and 40-kDa proteins discussed in the text. Protein standards are shown at the right with their molecular masses indicated in kilodaltons.

    Techniques Used: Affinity Purification, Magnetic Beads, Incubation, Infection, Binding Assay, Activity Assay, SDS Page, Silver Staining, Flow Cytometry, Mass Spectrometry, Expressing, Recombinant

    23) Product Images from "NDRG1 facilitates the replication and persistence of Kaposi’s sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA"

    Article Title: NDRG1 facilitates the replication and persistence of Kaposi’s sarcoma-associated herpesvirus by interacting with the DNA polymerase clamp PCNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007628

    NDRG1 directly interacts with PCNA. (A)Schematic procedure for purification and identification of NDRG1 binding proteins via TAP assay (left). Plasmid expressing Strep-Flag-tagged NDRG1 was stable transfected into KSHV positive iSLK.RGB cells. The equivalent empty vector was stable transfected as a control. Cell lysates were subjected to affinity purification with streptavidin beads, followed by IP with flag M2 beads. The purified elutes were resolved by SDS-PAGE and visualized with silver staining (right), and were also analyzed by MS. (B) Pathway pie chart showing classified and predicted functions of MS identified proteins. (C) Co-IP of NDRG1 and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-NDRG1 and anti-PCNA antibodies. (D) In vitro interaction between NDRG1 and PCNA via GST pull down assay. Purified GST, and GST-fused full length NDRG1 were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (E) NDRG1 colocalized with PCNA in the nucleus. Cells were fixed and probed with rabbit antibody against NDRG1 and mouse antibody against PCNA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (red), goat anti-mouse IgG conjugated with Alexa Fluor 488 (green), DAPI (blue). Scale bars represent 5μm.
    Figure Legend Snippet: NDRG1 directly interacts with PCNA. (A)Schematic procedure for purification and identification of NDRG1 binding proteins via TAP assay (left). Plasmid expressing Strep-Flag-tagged NDRG1 was stable transfected into KSHV positive iSLK.RGB cells. The equivalent empty vector was stable transfected as a control. Cell lysates were subjected to affinity purification with streptavidin beads, followed by IP with flag M2 beads. The purified elutes were resolved by SDS-PAGE and visualized with silver staining (right), and were also analyzed by MS. (B) Pathway pie chart showing classified and predicted functions of MS identified proteins. (C) Co-IP of NDRG1 and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-NDRG1 and anti-PCNA antibodies. (D) In vitro interaction between NDRG1 and PCNA via GST pull down assay. Purified GST, and GST-fused full length NDRG1 were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (E) NDRG1 colocalized with PCNA in the nucleus. Cells were fixed and probed with rabbit antibody against NDRG1 and mouse antibody against PCNA, followed by incubation with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (red), goat anti-mouse IgG conjugated with Alexa Fluor 488 (green), DAPI (blue). Scale bars represent 5μm.

    Techniques Used: Purification, Binding Assay, Plasmid Preparation, Expressing, Transfection, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, In Vitro, Pull Down Assay, Staining, Incubation

    NDRG1 forms a complex with LANA and PCNA. (A) LANA colocalized with NDRG1 and PCNA in the nucleus. Cells were fixed and probed with rat antibody against LANA, mouse antibody against PCNA, and rabbit antibody against NDRG1, followed by incubation with goat anti-rat IgG conjugated with Alexa Fluor 488 (green), goat anti-mouse IgG conjugated with Alexa Fluor 555 (red), goat anti-rabbit IgG conjugated with Alexa Fluor 680 (purple). Scale bars represent 5μm. (B) Co-IP of endogenous LANA, NDRG1, and PCNA in BCBL1 cells. Cell lysates were subjected to IP with anti-LANA mouse monoclonal antibody(1B5) or mouse IgG controls. Purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1, and anti-PCNA antibodies. (C) Co-IP of LANA, NDRG1, and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA and HA-LANA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. (D) Co-IP of LANA, NDRG1, and PCNA in HEK293T-Strep-Flag-LANA cells. pCDNA3.1-NDRG1 or pCDNA3.1-vector was transfected into cells. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. Tubulin was performed as the loading control of input samples, and IgG was used as the loading control of M2-Flag beads. (E) In vitro interaction between LANA and PCNA via GST pull down assay. Purified GST, and GST-fused LANA-N (1–340 aa) and GST-fused LANA-C (1022–1162 aa) beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (F) In vitro interaction between LANA and NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent IVT–NDRG1, and pulled down proteins were subjected to western blotting detection (upper panel). (G) In vitro interaction between LANA and PCNA in the presence of NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were incubated with equivalent IVT-PCNA and IVT–NDRG1, and pulled down proteins were subjected to western blotting detection.
    Figure Legend Snippet: NDRG1 forms a complex with LANA and PCNA. (A) LANA colocalized with NDRG1 and PCNA in the nucleus. Cells were fixed and probed with rat antibody against LANA, mouse antibody against PCNA, and rabbit antibody against NDRG1, followed by incubation with goat anti-rat IgG conjugated with Alexa Fluor 488 (green), goat anti-mouse IgG conjugated with Alexa Fluor 555 (red), goat anti-rabbit IgG conjugated with Alexa Fluor 680 (purple). Scale bars represent 5μm. (B) Co-IP of endogenous LANA, NDRG1, and PCNA in BCBL1 cells. Cell lysates were subjected to IP with anti-LANA mouse monoclonal antibody(1B5) or mouse IgG controls. Purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1, and anti-PCNA antibodies. (C) Co-IP of LANA, NDRG1, and PCNA in HEK293T cells. Strep-Flag-tagged NDRG1 was transfected into cells along with pCDNA3.1-PCNA and HA-LANA or empty vector controls. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. (D) Co-IP of LANA, NDRG1, and PCNA in HEK293T-Strep-Flag-LANA cells. pCDNA3.1-NDRG1 or pCDNA3.1-vector was transfected into cells. After affinity purification with M2-Flag beads, the purified proteins along with input samples were detected by western blotting with anti-LANA, anti-NDRG1 and anti-PCNA antibodies. Tubulin was performed as the loading control of input samples, and IgG was used as the loading control of M2-Flag beads. (E) In vitro interaction between LANA and PCNA via GST pull down assay. Purified GST, and GST-fused LANA-N (1–340 aa) and GST-fused LANA-C (1022–1162 aa) beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent in vitro translated IVT–PCNA, and pulled down proteins were subjected to western blotting detection (upper panel). (F) In vitro interaction between LANA and NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were subjected to SDS-PAGE and Coomassie Blue staining (lower panel). Purified beads were incubated with equivalent IVT–NDRG1, and pulled down proteins were subjected to western blotting detection (upper panel). (G) In vitro interaction between LANA and PCNA in the presence of NDRG1 via GST pull down assay. Purified GST, and GST-fused LANA-N and GST-fused LANA-C beads were incubated with equivalent IVT-PCNA and IVT–NDRG1, and pulled down proteins were subjected to western blotting detection.

    Techniques Used: Incubation, Co-Immunoprecipitation Assay, Purification, Western Blot, Transfection, Plasmid Preparation, Affinity Purification, In Vitro, Pull Down Assay, SDS Page, Staining

    24) Product Images from "Queuosine modification protects cognate tRNAs against ribonuclease cleavage"

    Article Title: Queuosine modification protects cognate tRNAs against ribonuclease cleavage

    Journal: RNA

    doi: 10.1261/rna.067033.118

    Q modification inhibits angiogenin cleavage in vitro. ( A ) Northern blot analysis of total RNAs isolated from 0Q, 100Q HEK293T cells probed against tRNA His , tRNA Asn , tRNA Asp , and tRNA Tyr  separated by APB-containing gels. Q, Q*, and G indicate tRNA with Q34, glycosylated Q34 and G34, respectively. ( B ) Appearance of Q modification for tRNA Asn  starting from the addition of queuine to HEK293T 0Q cells. Q and G indicate tRNA with Q34 and G34, respectively. ( C , D ) Comparative amount of angiogenin cleavage products upon varying the angiogenin concentration ( C ) or reaction time ( D ) of total tRNA isolated from 0Q and 100Q HEK293T cells. tRNAs were 5′  32 P-labeled, so the products can be identified by size. Comparison includes either only tRNA halfmer products corresponding to cleavage in the anticodon loop or all fragments derived from cleavage anywhere in the tRNA body. ( E ) Northern blot analysis of tRNA cleavage by angiogenin separated by APB-containing gels using the tRNA His  probe on the  left  and tRNA Asn  probe on the  right . Northern blot detected both 5′ and 3′ cleavage product in the anticodon loop; these halfmers in the 0Q sample are indicated by connecting lines on the  right , and the size of the products is shown in parentheses. The product near the full-length likely corresponds to angiogenin cleavage of the 3′CCA tail; these are indicated by an arrow and -CCA on the  right . Q-containing tRNA fragments are shifted in the 100Q sample.
    Figure Legend Snippet: Q modification inhibits angiogenin cleavage in vitro. ( A ) Northern blot analysis of total RNAs isolated from 0Q, 100Q HEK293T cells probed against tRNA His , tRNA Asn , tRNA Asp , and tRNA Tyr separated by APB-containing gels. Q, Q*, and G indicate tRNA with Q34, glycosylated Q34 and G34, respectively. ( B ) Appearance of Q modification for tRNA Asn starting from the addition of queuine to HEK293T 0Q cells. Q and G indicate tRNA with Q34 and G34, respectively. ( C , D ) Comparative amount of angiogenin cleavage products upon varying the angiogenin concentration ( C ) or reaction time ( D ) of total tRNA isolated from 0Q and 100Q HEK293T cells. tRNAs were 5′ 32 P-labeled, so the products can be identified by size. Comparison includes either only tRNA halfmer products corresponding to cleavage in the anticodon loop or all fragments derived from cleavage anywhere in the tRNA body. ( E ) Northern blot analysis of tRNA cleavage by angiogenin separated by APB-containing gels using the tRNA His probe on the left and tRNA Asn probe on the right . Northern blot detected both 5′ and 3′ cleavage product in the anticodon loop; these halfmers in the 0Q sample are indicated by connecting lines on the right , and the size of the products is shown in parentheses. The product near the full-length likely corresponds to angiogenin cleavage of the 3′CCA tail; these are indicated by an arrow and -CCA on the right . Q-containing tRNA fragments are shifted in the 100Q sample.

    Techniques Used: Modification, In Vitro, Northern Blot, Isolation, Concentration Assay, Labeling, Derivative Assay

    25) Product Images from "Discovery and Biological Characterization of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors"

    Article Title: Discovery and Biological Characterization of Potent Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors

    Journal: FEBS letters

    doi: 10.1002/1873-3468.12497

    Viability studies of specific human Bcl-2 family engineered cell lines after dosing with Mcl-1 inhibitors. (A,B) a panel of re-engineered BCR-ABL + B-ALL Cells modified to have the indicated anti-apoptotic exogenous Human Bcl-2 family member or DKO (double Bax,Bak knockout), TKO (triple Bax,Bak,Mcl-1 knockout) dosed with (A) compound 4 (B) compound 5.
    Figure Legend Snippet: Viability studies of specific human Bcl-2 family engineered cell lines after dosing with Mcl-1 inhibitors. (A,B) a panel of re-engineered BCR-ABL + B-ALL Cells modified to have the indicated anti-apoptotic exogenous Human Bcl-2 family member or DKO (double Bax,Bak knockout), TKO (triple Bax,Bak,Mcl-1 knockout) dosed with (A) compound 4 (B) compound 5.

    Techniques Used: Modification, Knock-Out

    Co-IP of Mcl-1 and Bim (A), or Bcl-xl and Bim (B) treated with three times the GI 50 from the proliferation assay in H929 cells, compound 4 (4.2 μM) and 5 (6.0 μM). The vehicle control, V , was treated with 0.1% DMSO.
    Figure Legend Snippet: Co-IP of Mcl-1 and Bim (A), or Bcl-xl and Bim (B) treated with three times the GI 50 from the proliferation assay in H929 cells, compound 4 (4.2 μM) and 5 (6.0 μM). The vehicle control, V , was treated with 0.1% DMSO.

    Techniques Used: Co-Immunoprecipitation Assay, Proliferation Assay

    Mitochondrial Depolarization studies. BH3 profiling with BAD (green) a Bcl-2,Bcl- x L binding peptide, MS-1 (red) a Mcl-1 selective binding peptide, HRK (magenta) a Bcl- x L selective binding peptide, and Bim (blue) a pan anti apoptotic (e.g. Bcl-2,Bcl- x L and, Mcl-1) binding peptide and with compound 4 (orange) and 5 (black) in (A) NCI H929 (B) K562 cells. (C) Comparison of cytochrome c release after dosing with the MS-1 peptide and compound 4 in a panel of Multiple Myeloma (MM) and Acute Myeloid Leukemia (AML) cell lines. (D) IC 50 values from a three day cell viability study after dosing compound 4 and 5 in a panel of AML and MM cell lines.
    Figure Legend Snippet: Mitochondrial Depolarization studies. BH3 profiling with BAD (green) a Bcl-2,Bcl- x L binding peptide, MS-1 (red) a Mcl-1 selective binding peptide, HRK (magenta) a Bcl- x L selective binding peptide, and Bim (blue) a pan anti apoptotic (e.g. Bcl-2,Bcl- x L and, Mcl-1) binding peptide and with compound 4 (orange) and 5 (black) in (A) NCI H929 (B) K562 cells. (C) Comparison of cytochrome c release after dosing with the MS-1 peptide and compound 4 in a panel of Multiple Myeloma (MM) and Acute Myeloid Leukemia (AML) cell lines. (D) IC 50 values from a three day cell viability study after dosing compound 4 and 5 in a panel of AML and MM cell lines.

    Techniques Used: Binding Assay, Mass Spectrometry

    Compound activity in freshly isolated patient samples. (A,B) Multiple Myeloma patient sample. (A) CD138 + cells were isolated from the bone marrow of a myeloma patient and treated with compound 5 for 24 h. Protein lysates were subjected to co-immunoprecipitation with monoclonal mouse anti-Mcl-1, anti-Bcl-x L , and monoclonal hamster anti-Bcl-2 antibodies. Resulting protein complexes were subjected to Western blot analysis using rabbit antibodies against Mcl-1, Bcl-x L , Bcl-2, and Bim. (B) Ficoll isolated buffy coat cells were treated with the indicated concentrations of compound 4 for 24 h. Apoptosis was determined by staining with antibodies against CD38, CD45, and Annexin-V-FITC. The percent annexin/PI positive cells represent the specific cell death after subtracting the spontaneous cell death. (C) Dose dependent viability decrease in Acute Myeloid Leukemia (AML) patient sample with 4 (triangle) and 5 (open circle).
    Figure Legend Snippet: Compound activity in freshly isolated patient samples. (A,B) Multiple Myeloma patient sample. (A) CD138 + cells were isolated from the bone marrow of a myeloma patient and treated with compound 5 for 24 h. Protein lysates were subjected to co-immunoprecipitation with monoclonal mouse anti-Mcl-1, anti-Bcl-x L , and monoclonal hamster anti-Bcl-2 antibodies. Resulting protein complexes were subjected to Western blot analysis using rabbit antibodies against Mcl-1, Bcl-x L , Bcl-2, and Bim. (B) Ficoll isolated buffy coat cells were treated with the indicated concentrations of compound 4 for 24 h. Apoptosis was determined by staining with antibodies against CD38, CD45, and Annexin-V-FITC. The percent annexin/PI positive cells represent the specific cell death after subtracting the spontaneous cell death. (C) Dose dependent viability decrease in Acute Myeloid Leukemia (AML) patient sample with 4 (triangle) and 5 (open circle).

    Techniques Used: Activity Assay, Isolation, Immunoprecipitation, Western Blot, Staining

    26) Product Images from "A novel Rac-dependent checkpoint in B cell development controls entry into the splenic white pulp and cell survival"

    Article Title: A novel Rac-dependent checkpoint in B cell development controls entry into the splenic white pulp and cell survival

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20091489

    Vav family proteins are not required for entry into splenic white pulp. (A) Contour plots of splenocytes from either WT (B10.BR) or Vav1 −/− Vav2 −/− Vav3 −/− (VavTKO) mice showing separation of B220 + cells into immature (CD93 + ) and mature (CD93 − ) B cells. Note that the VavTKO mice are on a B10.BR background and are therefore compared with WT B10.BR mice. The immature cells were further gated on IgM + cells and separated according to the expression of IgD and CD23 into T0 (IgD − CD23 − ), T1 IgD + (IgD + CD23 − ), and T2 (IgD + CD23 + ) subsets. Mature (CD93 − ) cells were separated according to expression of IgM and CD23 into MRF (IgM +/− CD23 + ) and MZ (IgM + CD23 − ) subsets (not depicted). Numbers show percentages of cells falling into gates or quadrants. (B) Mean (±SEM) number of splenic T0, T1 IgD + , T2, and MRF B cells in WT ( n = 5), Rac1 B Rac2 −/− ( n = 7), B10.BR, and VavTKO mice ( n = 7). (C) Mean (±SEM) migration in a Transwell assay of T0, T1 IgD + , and T2 splenic B cells from WT (B10.BR) or VavTKO mice in response to the indicated chemokines ( n = 6). (D) Images showing immunofluorescence staining of sections from spleens of mice into which WT (B10.BR) or VavTKO (IgM b ) T0 B cells had been transferred 24 h earlier. Staining for IgM b (green) identifies transferred T0 B cells, and MadCAM-1 (red) defines the edges of the white pulp. Bar, 150 µm. The graph shows the mean (±SEM) ratio of transferred IgM b+ T0 B cells ending up in white relative to red splenic pulp 24 h after transfer ( n = 4). *, P
    Figure Legend Snippet: Vav family proteins are not required for entry into splenic white pulp. (A) Contour plots of splenocytes from either WT (B10.BR) or Vav1 −/− Vav2 −/− Vav3 −/− (VavTKO) mice showing separation of B220 + cells into immature (CD93 + ) and mature (CD93 − ) B cells. Note that the VavTKO mice are on a B10.BR background and are therefore compared with WT B10.BR mice. The immature cells were further gated on IgM + cells and separated according to the expression of IgD and CD23 into T0 (IgD − CD23 − ), T1 IgD + (IgD + CD23 − ), and T2 (IgD + CD23 + ) subsets. Mature (CD93 − ) cells were separated according to expression of IgM and CD23 into MRF (IgM +/− CD23 + ) and MZ (IgM + CD23 − ) subsets (not depicted). Numbers show percentages of cells falling into gates or quadrants. (B) Mean (±SEM) number of splenic T0, T1 IgD + , T2, and MRF B cells in WT ( n = 5), Rac1 B Rac2 −/− ( n = 7), B10.BR, and VavTKO mice ( n = 7). (C) Mean (±SEM) migration in a Transwell assay of T0, T1 IgD + , and T2 splenic B cells from WT (B10.BR) or VavTKO mice in response to the indicated chemokines ( n = 6). (D) Images showing immunofluorescence staining of sections from spleens of mice into which WT (B10.BR) or VavTKO (IgM b ) T0 B cells had been transferred 24 h earlier. Staining for IgM b (green) identifies transferred T0 B cells, and MadCAM-1 (red) defines the edges of the white pulp. Bar, 150 µm. The graph shows the mean (±SEM) ratio of transferred IgM b+ T0 B cells ending up in white relative to red splenic pulp 24 h after transfer ( n = 4). *, P

    Techniques Used: Mouse Assay, Expressing, Migration, Transwell Assay, Immunofluorescence, Staining

    Pertussis toxin blocks entry of T0 splenic B cells into the white pulp, as well as their survival. (A) Mean (±SEM) ratio of transferred IgM b+ T0 B cells ending up in white relative to red splenic pulp at 24 h after transfers, as described in Fig. 4 (A–C) . Transferred T0 cells were from WT or Rac1 B Rac2 −/− mice. In some transfers of WT T0 B cells, the mice were pretreated with anti–LFA-1 and anti-α4 blocking antibodies (antiintegrin mAb), or the cells were treated with pertussis toxin ( n = 4–6). (B and D) Contour plots show expression of IgD and CD23 on B220 + IgM b+ splenocytes from 129S8 (IgM a+ ) mice into which (B) WT or (D) Rac1 B Rac2 −/− (T0 B cells (both IgM b+ ) had been transferred 24 h earlier and had been either pretreated with pertussis toxin or an oligomer of the B subunit of pertussis toxin (Oligo B). Oligo B controlled for any effects of pertussis toxin independent of the ADP-ribosylation activity of the A subunit, which inactivates Gαi-coupled GPCRs. Staining of input cells before transfer is shown for comparison. Numbers indicate percentages of cells falling into quadrants. (C and E) Graphs show the mean (±SEM) percent recovery of transitional B cells in the spleens of 129S8 mice 24 h after transfer of WT (C; n = 4–5) or Rac1 B Rac2 −/− (E; n = 6–7) T0 B cells pretreated with Oligo B or pertussis toxin. Recovery of total transitional cells is shown as well as subdivision of these into T0, T1 IgD + , and T2 cells. *, P
    Figure Legend Snippet: Pertussis toxin blocks entry of T0 splenic B cells into the white pulp, as well as their survival. (A) Mean (±SEM) ratio of transferred IgM b+ T0 B cells ending up in white relative to red splenic pulp at 24 h after transfers, as described in Fig. 4 (A–C) . Transferred T0 cells were from WT or Rac1 B Rac2 −/− mice. In some transfers of WT T0 B cells, the mice were pretreated with anti–LFA-1 and anti-α4 blocking antibodies (antiintegrin mAb), or the cells were treated with pertussis toxin ( n = 4–6). (B and D) Contour plots show expression of IgD and CD23 on B220 + IgM b+ splenocytes from 129S8 (IgM a+ ) mice into which (B) WT or (D) Rac1 B Rac2 −/− (T0 B cells (both IgM b+ ) had been transferred 24 h earlier and had been either pretreated with pertussis toxin or an oligomer of the B subunit of pertussis toxin (Oligo B). Oligo B controlled for any effects of pertussis toxin independent of the ADP-ribosylation activity of the A subunit, which inactivates Gαi-coupled GPCRs. Staining of input cells before transfer is shown for comparison. Numbers indicate percentages of cells falling into quadrants. (C and E) Graphs show the mean (±SEM) percent recovery of transitional B cells in the spleens of 129S8 mice 24 h after transfer of WT (C; n = 4–5) or Rac1 B Rac2 −/− (E; n = 6–7) T0 B cells pretreated with Oligo B or pertussis toxin. Recovery of total transitional cells is shown as well as subdivision of these into T0, T1 IgD + , and T2 cells. *, P

    Techniques Used: Mouse Assay, Blocking Assay, Expressing, Activity Assay, Staining

    Accumulation of Rac-deficient immature B cells in the blood may be caused by defective CXCR4 signaling. (A) Splenic T0 B cells from mice of the indicated genotypes (IgM b Ly5.2 + ) mixed with splenic T0 B cells from 129S8 (IgM a Ly5.2 + ) mice were injected into B6.SJL (IgM b Ly5.1 + ) mice. Graph shows the mean (±SEM) IgM b Ly5.2 + to IgM a Ly5.2 + ratio of B cells in the blood, spleen, and bone marrow 4 h after transfer, normalized to the input ratio ( n = 5–17). (B) Mean (±SEM) number of IgM b+ cells recovered from the blood, spleen, and bone marrow of B6.SJL mice into which WT splenic T0 B cells had been transferred 4 h earlier in the absence or presence of the CXCR4 inhibitor AMD3100 ( n = 4). (C) Mean (±SEM) migration of immature bone marrow or splenic T0 cells from mice of the indicated genotypes in a Transwell assay in response to CXCL12. Wells were coated or not (−) with VCAM-1 ( n = 6). *, P
    Figure Legend Snippet: Accumulation of Rac-deficient immature B cells in the blood may be caused by defective CXCR4 signaling. (A) Splenic T0 B cells from mice of the indicated genotypes (IgM b Ly5.2 + ) mixed with splenic T0 B cells from 129S8 (IgM a Ly5.2 + ) mice were injected into B6.SJL (IgM b Ly5.1 + ) mice. Graph shows the mean (±SEM) IgM b Ly5.2 + to IgM a Ly5.2 + ratio of B cells in the blood, spleen, and bone marrow 4 h after transfer, normalized to the input ratio ( n = 5–17). (B) Mean (±SEM) number of IgM b+ cells recovered from the blood, spleen, and bone marrow of B6.SJL mice into which WT splenic T0 B cells had been transferred 4 h earlier in the absence or presence of the CXCR4 inhibitor AMD3100 ( n = 4). (C) Mean (±SEM) migration of immature bone marrow or splenic T0 cells from mice of the indicated genotypes in a Transwell assay in response to CXCL12. Wells were coated or not (−) with VCAM-1 ( n = 6). *, P

    Techniques Used: Mouse Assay, Injection, Migration, Transwell Assay

    Transitional B cells deficient in both Rac1 and Rac2 fail to enter the white pulp of the spleen. (A and B) Contour plots show IgD and CD23 expression on B220 + IgM b+ cells from 129S8 (IgM a ) mice into which splenic T0 B cells from WT or Rac1 B Rac2 −/− (IgM b ) mice had been transferred (A) 4 h or (B) 24 h earlier. The input cells before transfer are shown for comparison. Numbers indicate percentages of cells falling into quadrants. (C) Images showing immunofluorescence staining of sections from spleens of mice into which WT or Rac1 B Rac2 −/− (IgM b ) T0 B cells had been transferred 4 or 24 h earlier. Staining for IgM b (green) identifies transferred T0 B cells, and MadCAM-1 (red) defines the edges of the white pulp. (right) Transferred WT MRF B cells for comparison. Bar, 150 µm. (D) Mean (±SEM) ratio of transferred IgM b+ T0 or MRF B cells ending up in white relative to red splenic pulp at 4 or 24 h after transfer, quantitated using sections such as those shown in C ( n = 4–6). (E) Splenic T0 B cells from mice of the indicated genotypes (IgM b Ly5.2 + ) were injected into 129S8 (IgM a Ly5.2 + ) mice. Graph shows the mean (±SEM) percent recovery of transitional B cells in the spleen 24 h after transfer of T0 cells of the indicated genotype. Recovery of total transitional cells is shown, as well as subdivision of these into T0, T1 IgD + , and T2 cells ( n = 5–8). *, P
    Figure Legend Snippet: Transitional B cells deficient in both Rac1 and Rac2 fail to enter the white pulp of the spleen. (A and B) Contour plots show IgD and CD23 expression on B220 + IgM b+ cells from 129S8 (IgM a ) mice into which splenic T0 B cells from WT or Rac1 B Rac2 −/− (IgM b ) mice had been transferred (A) 4 h or (B) 24 h earlier. The input cells before transfer are shown for comparison. Numbers indicate percentages of cells falling into quadrants. (C) Images showing immunofluorescence staining of sections from spleens of mice into which WT or Rac1 B Rac2 −/− (IgM b ) T0 B cells had been transferred 4 or 24 h earlier. Staining for IgM b (green) identifies transferred T0 B cells, and MadCAM-1 (red) defines the edges of the white pulp. (right) Transferred WT MRF B cells for comparison. Bar, 150 µm. (D) Mean (±SEM) ratio of transferred IgM b+ T0 or MRF B cells ending up in white relative to red splenic pulp at 4 or 24 h after transfer, quantitated using sections such as those shown in C ( n = 4–6). (E) Splenic T0 B cells from mice of the indicated genotypes (IgM b Ly5.2 + ) were injected into 129S8 (IgM a Ly5.2 + ) mice. Graph shows the mean (±SEM) percent recovery of transitional B cells in the spleen 24 h after transfer of T0 cells of the indicated genotype. Recovery of total transitional cells is shown, as well as subdivision of these into T0, T1 IgD + , and T2 cells ( n = 5–8). *, P

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Staining, Injection

    27) Product Images from "rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences"

    Article Title: rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04370-x

    rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 corrects splicing alterations caused by CCUG repeats. a Upper panel, RT-PCR analysis of alternative splicing of the mouse chloride channel Clcn1 exon 6B minigene co-transfected in C2C12 mouse muscle cells with a plasmid expressing either 960 CUG repeats or 1000 CCUG repeats and a vector expressing either rbFOX1 or MBNL1. Lower panel, quantification of Clcn1 exon 6B inclusion. b As in a but with TNNT2 (cTNT) exon 5 minigene. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing

    Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f Quantification of the co-localization of CUG or CCUG RNA foci with candidate proteins in transfected C2C12 cells. Error bars indicate s.e.m. of three independent experiments. Representative images are presented in Supplementary Fig. 1
    Figure Legend Snippet: Identification of proteins specifically associated with expanded CCUG repeats. a UV-crosslinking binding assays of 20 µg of nuclear extract from C2C12 muscle cells differentiated four days incubated with 30,000 CPM of uniformly [αP 32 ] internally labeled in vitro transcribed RNAs containing 30 CUG or CCUG repeats. b Silver staining of proteins extracted from 1 mg of mouse brain and captured on streptavidin resin coupled to biotinylated RNA containing 30 CUG or CCUG repeats. c Western blotting against either rbFox1 or Mbnl1 on mouse brain proteins captured by RNA-column containing either 30 CUG or 30 CCUG repeats. d RNA FISH against CCUG repeats coupled to immunofluorescence against Mbnl1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. e RNA FISH against CCUG repeats coupled to immunofluorescence against rbFox1 on differentiated C2C12 cells transfected with a plasmid expressing either 960 CUG or 1000 CCUG repeats. Scale bars, 10 µm. Nuclei were counterstained with DAPI. f Quantification of the co-localization of CUG or CCUG RNA foci with candidate proteins in transfected C2C12 cells. Error bars indicate s.e.m. of three independent experiments. Representative images are presented in Supplementary Fig. 1

    Techniques Used: Binding Assay, Incubation, Labeling, In Vitro, Silver Staining, Western Blot, Fluorescence In Situ Hybridization, Immunofluorescence, Transfection, Plasmid Preparation, Expressing

    rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p
    Figure Legend Snippet: rbFOX1 is not sequestered within CCUG RNA foci. a Time course quantification of photoconverted spot of dendra2-rbFOX1 in COS7 cells co-transfected with a plasmid expressing dendra2-rbFOX1 and a plasmid expressing either no repeats (CTL), 960 CUG or 1000 CCUG repeats. Each data point is the average of 7 spot. b As in a but with dendra2-MBNL1. c Upper panel, RT-PCR analysis of RNA extracted from two days differentiated C2C12 cells co-transfected with a minigene expressing the exon 9 of the mitochondrial ATP synthase gamma-subunit gene and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of exon 9 inclusion of transfected ATP5C1 minigene. d Upper panel, RT-PCR analysis of endogenous Fmnl3 exon 26 alternative splicing from GFP-FACS sorted C2C12 cells differentiated two days and co-transfected with a plasmid expressing eGFP and either with a plasmid expressing rbFOX1, MBNL1, 960 CUG repeats or 1000 CCUG repeats or with a siRNA directed against rbFox1 or Mbnl1 . Lower panel, quantification of Fmnl3 exon 26 inclusion. e–g RT-PCR analysis (left panel) and quantification (right panel) of alternative splicing of FMNL3, ENAH , and ECT2 performed on total RNA extracted from adult skeletal muscle of control or DM2 individuals. Error bars indicate s.e.m. of three independent experiments. Student’s t -test, asterisk (*) indicates p

    Techniques Used: Transfection, Plasmid Preparation, Expressing, CTL Assay, Reverse Transcription Polymerase Chain Reaction, FACS

    28) Product Images from "Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133"

    Article Title: Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-186

    DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).
    Figure Legend Snippet: DNA affinity assay of the hupS / Npun_R0367 promoter region from Nostoc punctiforme ATCC 29133 and the hupS / asr0389 promoter region from Nostoc sp. strain PCC 7120 and total protein extract from respective strain . SDS-PAGE of proteins interacting with (A) the hupS / Npun_R0367 promoter region from N. punctiforme and (B) the hupS / asr0389 promoter region from Nostoc PCC 7120 from DNA-protein affinity assays. Lanes: M) protein molecular weight marker; 1) Total protein extract, 2) DNA-free negative control, 3) hupS / Npun_R0367 or hupS / asr0389 promoter region respectively. The unlabelled bands on the gel, present in both negative controls and samples, correspond to identified peptides either from unspecific binding, e.g. phycobilisome linker polypeptide (weak bands), artifacts from the experimental procedure, e.g. streptavidin (strongest band) or peptides with too low concentration to be identified (*).

    Techniques Used: Periodic Counter-current Chromatography, SDS Page, Molecular Weight, Marker, Negative Control, Binding Assay, Concentration Assay

    Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.
    Figure Legend Snippet: Transcript levels of the ORFs upstream of the hyp -genes in Nostoc sp. strain PCC 7120 after nitrogen depletion . Agarose gels showing the amplified PCR products using cDNA prepared RNA from Nostoc PCC 7120 cultures 0, 24, 48 and 72 hours after nitrogen depletion as well as isolated heterocysts 48 hours after nitrogen depletion. The tested genes are the hydrogenase and ribosome structural genes hupS and 23S , the hyp -genes hypC and hypF and the ORFs upstream of the hyp -genes ( asr0689 , asr0690 , alr0691 , alr0691 and alr0693 ). All DNA fragments were amplified with PCR using 30 cycles, except for nifD and 23S where 25 and 15 cycles were used, respectively. Negative (-) and positive controls (+) for the PCR reactions are shown.

    Techniques Used: Periodic Counter-current Chromatography, Amplification, Polymerase Chain Reaction, Isolation

    Electrophoretic mobility shift assay of the hupS/ Npun_R0367 promoter region in Nostoc punctiforme ATCC 29133 . Electrophoretic mobility shift assay showing specific binding of purified CalA to the N. punctiforme hupS upstream region. C1 - 308 bp control fragment, C2 - 1350 bp control fragment, P hupS - 558 bp hupS immediate upstream region fragment. 100 ng of each fragment and increasing amounts (see label for each lane) of purified histidine-tagged CalA (His-CalA) from Nostoc PCC 7120 were used in the reaction mixtures.
    Figure Legend Snippet: Electrophoretic mobility shift assay of the hupS/ Npun_R0367 promoter region in Nostoc punctiforme ATCC 29133 . Electrophoretic mobility shift assay showing specific binding of purified CalA to the N. punctiforme hupS upstream region. C1 - 308 bp control fragment, C2 - 1350 bp control fragment, P hupS - 558 bp hupS immediate upstream region fragment. 100 ng of each fragment and increasing amounts (see label for each lane) of purified histidine-tagged CalA (His-CalA) from Nostoc PCC 7120 were used in the reaction mixtures.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Purification, Periodic Counter-current Chromatography

    Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.
    Figure Legend Snippet: Genomic arrangement of the ORFs upstream of the hyp -genes in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120 . (A) In the filamentous, heterocyst forming cyanobacterial strain N. punctiforme the five ORFs upstream of the hyp -genes are located upstream of the uptake hydrogenase structural genes, hupSL , and in between hupSL and the hyp -genes, hypFCDEAB . (B) The same genomic arrangement can be found in the filamentous, heterocyst forming cyanobacterial strain Nostoc PCC 7120. This genomic arrangement of the ORFs upstream of the hyp -genes seems to be conserved in filamentous cyanobacteria harboring an uptake hydrogenase [ 19 ] * indicates the 5' end of hupL (encoding the N-terminal end of HupL) as it is annotated in vegetative cells. The identified tsps upstream of hupSL [ 36 ], Npun_R0363 [ 27 ] and Npun_R0367 (this work) in ATCC 29133 and upstream of asr0689 , hypF and hypC in Nostoc PCC 7120 [ 19 ] are indicated by arrows.

    Techniques Used: Periodic Counter-current Chromatography

    29) Product Images from "Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format"

    Article Title: Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format

    Journal: Nucleic Acids Research

    doi:

    Schematic representation of the mutS chip assay. (I) Biotinylated reference strands (e.g. PCR products) are first addressed to individual test sites of the array using electronic biassing. (II) Cy3-labelled complementary test strands are ‘electronically’ hybridised to the reference strands, thereby generating heteroduplex DNA. (III) The Cy5mutS protein binds preferentially to mismatched heteroduplex DNA. Hybridisation and binding events are monitored by fluorescence scanning of the array.
    Figure Legend Snippet: Schematic representation of the mutS chip assay. (I) Biotinylated reference strands (e.g. PCR products) are first addressed to individual test sites of the array using electronic biassing. (II) Cy3-labelled complementary test strands are ‘electronically’ hybridised to the reference strands, thereby generating heteroduplex DNA. (III) The Cy5mutS protein binds preferentially to mismatched heteroduplex DNA. Hybridisation and binding events are monitored by fluorescence scanning of the array.

    Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, DNA Hybridization, Binding Assay, Fluorescence

    Parallel mutation scanning of p53 exons 8 and 9 from human cell lines. Biotinylated sense and antisense strands of the exons were generated by PCR using a wild-type p53 gene as template, and addressed row-by-row as follows. Lane 1, exon 8 antisense; lane 2, exon 8 sense; lane 3, exon 9 antisense; lane 4, exon 9 sense. Subsequently, the complementary PCR amplified strands from the indicated cell lines were addressed to generate heteroduplex DNA. ( A ) The Cy3 image of the array indicates even hybridisation. ( B ) Staining of the array with Cy5mutS revealed a mutation in both exons 8 and 9 of the cell line SW-480.
    Figure Legend Snippet: Parallel mutation scanning of p53 exons 8 and 9 from human cell lines. Biotinylated sense and antisense strands of the exons were generated by PCR using a wild-type p53 gene as template, and addressed row-by-row as follows. Lane 1, exon 8 antisense; lane 2, exon 8 sense; lane 3, exon 9 antisense; lane 4, exon 9 sense. Subsequently, the complementary PCR amplified strands from the indicated cell lines were addressed to generate heteroduplex DNA. ( A ) The Cy3 image of the array indicates even hybridisation. ( B ) Staining of the array with Cy5mutS revealed a mutation in both exons 8 and 9 of the cell line SW-480.

    Techniques Used: Mutagenesis, Generated, Polymerase Chain Reaction, Amplification, Hybridization, Staining

    30) Product Images from "lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer"

    Article Title: lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer

    Journal: Scientific Reports

    doi: 10.1038/srep10159

    lncRNA GAS5 interacts with the transcriptional activator YBX1. ( a ) Western blot detected the YBX1 in the GAS5 pull-down complex. GAS5-FL was the biotin labeled lncRNA GAS5, NS was the biotin labeled non-sense RNA with similar length to GAS5, NC was the lncRNA GAS5 without biotin label. ( b ) qRT-PCR confirmed that lncRNA GAS5 was accumulated in YBX1-precipatated protein sample. ( c ) The agarose gel electrophoresis graph showed PCR products of RIP. ( d ) YBX1 protein level was decreased with lncRNA GAS5 knock-down. ( e ) The YBX1 mRNA levels were not affected by lncRNA GAS5 knock-down. NC was the negative control sequence for siRNAs. ( f ) and ( g ) lncRNA GAS5 had effect on YBX1 protein turnover under the treatment of CHX at the indicate interval in HGC-27 cells ( f ) and in SGC-7901 cells ( g ). *, p
    Figure Legend Snippet: lncRNA GAS5 interacts with the transcriptional activator YBX1. ( a ) Western blot detected the YBX1 in the GAS5 pull-down complex. GAS5-FL was the biotin labeled lncRNA GAS5, NS was the biotin labeled non-sense RNA with similar length to GAS5, NC was the lncRNA GAS5 without biotin label. ( b ) qRT-PCR confirmed that lncRNA GAS5 was accumulated in YBX1-precipatated protein sample. ( c ) The agarose gel electrophoresis graph showed PCR products of RIP. ( d ) YBX1 protein level was decreased with lncRNA GAS5 knock-down. ( e ) The YBX1 mRNA levels were not affected by lncRNA GAS5 knock-down. NC was the negative control sequence for siRNAs. ( f ) and ( g ) lncRNA GAS5 had effect on YBX1 protein turnover under the treatment of CHX at the indicate interval in HGC-27 cells ( f ) and in SGC-7901 cells ( g ). *, p

    Techniques Used: Western Blot, Labeling, Quantitative RT-PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Sequencing

    LncRNA GAS5 mutant fails to arrest cell cycle at the G1 phase. ( a ) Western blot detected YBX1 from GAS5-DEL and GAS5-FL RNA pull-down complexes. GAS5-DEL was biotin labeled lncRNA GAS5 mutant with its exon 12 deletion. GAS5-FL was biotin labeled full length lncRNA GAS5. NS was the biotin labeled non-sense RNA with similar length to GAS5, NC was the lncRNA GAS5 without biotin label. ( b ) The protein level of p21 and YBX1 after transfection with GAS5-E, GAS5-D or GAS5-B plasmids. ( c ) and ( d ) The cell cycle alteration after transfecting GAS5-E, GAS5-D or GAS5-B plasmids in HGC-27 ( c ) and in SGC-7901 ( d ). GAS5-E was the lncRNA GAS5 expression plasmid, GAS5-D was the lncRNA GAS5 mutant with its exon 12 deleted plasmid, and GAS5-B was the lncRNA GAS5 blank empty plasmid. *, p
    Figure Legend Snippet: LncRNA GAS5 mutant fails to arrest cell cycle at the G1 phase. ( a ) Western blot detected YBX1 from GAS5-DEL and GAS5-FL RNA pull-down complexes. GAS5-DEL was biotin labeled lncRNA GAS5 mutant with its exon 12 deletion. GAS5-FL was biotin labeled full length lncRNA GAS5. NS was the biotin labeled non-sense RNA with similar length to GAS5, NC was the lncRNA GAS5 without biotin label. ( b ) The protein level of p21 and YBX1 after transfection with GAS5-E, GAS5-D or GAS5-B plasmids. ( c ) and ( d ) The cell cycle alteration after transfecting GAS5-E, GAS5-D or GAS5-B plasmids in HGC-27 ( c ) and in SGC-7901 ( d ). GAS5-E was the lncRNA GAS5 expression plasmid, GAS5-D was the lncRNA GAS5 mutant with its exon 12 deleted plasmid, and GAS5-B was the lncRNA GAS5 blank empty plasmid. *, p

    Techniques Used: Mutagenesis, Western Blot, Labeling, Transfection, Expressing, Plasmid Preparation

    31) Product Images from "Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer"

    Article Title: Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-374

    Over-expression of miR-145 in SW620 cell line affects cell morphology and proliferation . (A) The genomic region surrounding the miR-145 gene was PCR-amplified and cloned into pSilencer 4.1 under control of the CMV promoter. Mature miR-145 was detected by Northern analysis in a pooled population of SW620 cells following transfection. U6 snRNA was used as a loading control. (B) A major distinguishing feature of the cell population over-expressing miR-145 was the change in cell morphology from the round single cells of SW620 to elongated cells with extended processes typical of fibroblast-like cells. (C) The miR-145-expressing SW620 cell population showed a two-fold increase in anchorage-independent growth when grown in the presence of serum and a greater than 50% increase in cell proliferation/metabolic activity when grown in the presence (solid bars) or absence (open bars) of serum. *** p
    Figure Legend Snippet: Over-expression of miR-145 in SW620 cell line affects cell morphology and proliferation . (A) The genomic region surrounding the miR-145 gene was PCR-amplified and cloned into pSilencer 4.1 under control of the CMV promoter. Mature miR-145 was detected by Northern analysis in a pooled population of SW620 cells following transfection. U6 snRNA was used as a loading control. (B) A major distinguishing feature of the cell population over-expressing miR-145 was the change in cell morphology from the round single cells of SW620 to elongated cells with extended processes typical of fibroblast-like cells. (C) The miR-145-expressing SW620 cell population showed a two-fold increase in anchorage-independent growth when grown in the presence of serum and a greater than 50% increase in cell proliferation/metabolic activity when grown in the presence (solid bars) or absence (open bars) of serum. *** p

    Techniques Used: Over Expression, Polymerase Chain Reaction, Amplification, Clone Assay, Northern Blot, Transfection, Expressing, Activity Assay

    32) Product Images from "The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain"

    Article Title: The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1468

    Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).
    Figure Legend Snippet: Nrd1 binds preferentially to CTD-Ser5P. ( a ) Binding to four repeat CTD peptides in vitro . Unmodified, Ser2P, Ser5P or Ser2P/Ser5P peptides were immobilized on streptavidin-conjugated magnetic beads and incubated with 5 µg of recombinant Nrd1 (rNrd1), 10 ng of TAP-purified yeast Nrd1 complex (yNRD1) or 500 µg of whole-cell extract from an Rtt103-hemagglutinin (HA) strain (YSB815). Bound proteins were eluted, separated by SDS-PAGE and detected by immunoblotting using either anti-Nrd1 or anti-HA antibodies. Recombinant Rtt103 also specifically bound to CTD-Ser2P (not shown). ( b ) Nrd1 6–151 was titrated with fluorescently labeled CTD-Ser5P (two repeats) and binding was measured by fluorescence anisotropy (black triangles; ref.-FAM, 5,6-carboxyfluorescein labeled reference). The same experiment was then done in the presence of competing unlabeled CTD-Ser2P (circles), CTDSer5P (white triangles) or CTD-Ser2P/Ser5P (diamonds). Equilibrium dissociation constants ( K d ) were calculated from the best fit to the data. ( c ) Nrd1 is associated with Ser5-phosphorylated Pol II in vivo . Nrd1 was purified via the TAP tag, and the phosphorylation status of the associated polymerase was monitored by immunoblotting using anti-CTD (8WG16), anti-Ser2P (H5), anti-Ser5P (H14) or an antibody that can recognize both Ser2P and Ser5P (B3) 9 . ( d ) Ctk1 kinase is not required for recruitment of Nrd1 to genes in vivo . Cross-linked chromatin was prepared from Nrd1-TAP–containing cells that were wild-type (WT) or deleted (Δctk1) for the CTK1 gene. Following precipitation with IgG agarose, chromatin was amplified with primers across the snR33 locus, as diagrammed below. Immunoprecipitated samples (IP) were compared against input chromatin (Input) and quantified (right). The upper band in each lane is the snR33 product and the lower band is a nontranscribed control region. Similar results were obtained for the PMA1 and ADH1 genes (not shown).

    Techniques Used: Binding Assay, In Vitro, Magnetic Beads, Incubation, Recombinant, Purification, SDS Page, Labeling, Fluorescence, In Vivo, Amplification, Immunoprecipitation

    33) Product Images from "Enrichment of meiotic recombination hotspot sequences by avidin capture technology"

    Article Title: Enrichment of meiotic recombination hotspot sequences by avidin capture technology

    Journal: Gene

    doi: 10.1016/j.gene.2012.12.042

    Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.
    Figure Legend Snippet: Capture of long dsDNA with oligo-Blue. (A) EcoRI fragment of pBluescript II sk (+), containing a 13-mer with a degree of degeneration similar to the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).
    Figure Legend Snippet: Capture and release of a fluorophore-conjugated oligonucleotide. Oligonuclotide oligo-3-FAM is labeled with fluorescein and contains the hotspot sequence for recombination. The numbers denote the sequential washes of streptavidin beads before and after treatment with EcoRI . Fraction 9 depicts the fluorescence in beads after the final wash (N=3; the image represents a representative example).

    Techniques Used: Labeling, Sequencing, Fluorescence

    Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.
    Figure Legend Snippet: Enrichment of a DNA containing the hotspot motif from a mixture of short, double-stranded DNA. (A) Synthetic dsDNA oligonucleotides: 100-mer containing the hotspot sequence for recombination; 50-mer not containing the hotspot sequence. (B) PCR-amplified samples of supernatants from streptavidin beads after loading with oligonucleotides (sample 1), sequential washes of beads (samples 2 – 9), and supernatant after treating beads with EcoRI (sample 10). (C) As described for “A” but now the 50-mer contains the hotspot sequence. (D) As described for “B.” Abbreviation: M, marker.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Marker

    34) Product Images from "Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: Implication of PARP-1, APEX-1 and ERK1/2 catalytic activities"

    Article Title: Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: Implication of PARP-1, APEX-1 and ERK1/2 catalytic activities

    Journal: Biochimica et Biophysica Acta. Molecular Cell Research

    doi: 10.1016/j.bbamcr.2010.04.011

    Purification of nuclear proteins binding to the SRE1-EBS-GRE elements of the human apoD promoter. (A) Nuclear extracts from normal growth (10% serum: +) or growth arrest (0.5% serum: −) conditions were incubated with either the biotinylated oligonucleotide bound to streptavidin beads (NE) or streptavidin beads alone (CTRL). The bound proteins were eluted and analyzed by SDS-PAGE and silver staining. The numbers and arrows on the gel indicate the excised bands. (B) Mass-spectrometry analysis of the eluted bands. Nuclear factors were identified by mass spectrometry (LC-MSMS) after tryptic digestion. STD: Standard molecular weight in kDa; this experiment was done in duplicate.
    Figure Legend Snippet: Purification of nuclear proteins binding to the SRE1-EBS-GRE elements of the human apoD promoter. (A) Nuclear extracts from normal growth (10% serum: +) or growth arrest (0.5% serum: −) conditions were incubated with either the biotinylated oligonucleotide bound to streptavidin beads (NE) or streptavidin beads alone (CTRL). The bound proteins were eluted and analyzed by SDS-PAGE and silver staining. The numbers and arrows on the gel indicate the excised bands. (B) Mass-spectrometry analysis of the eluted bands. Nuclear factors were identified by mass spectrometry (LC-MSMS) after tryptic digestion. STD: Standard molecular weight in kDa; this experiment was done in duplicate.

    Techniques Used: Purification, Binding Assay, Incubation, SDS Page, Silver Staining, Mass Spectrometry, Molecular Weight

    35) Product Images from "Biochemical characterization of the helicase Sen1 provides new insights into the mechanisms of non-coding transcription termination"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1230

    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.
    Figure Legend 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.

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

    36) Product Images from "Particle-Based Microfluidic Quartz Crystal Microbalance (QCM) Biosensing Utilizing Mass Amplification and Magnetic Bead Convection"

    Article Title: Particle-Based Microfluidic Quartz Crystal Microbalance (QCM) Biosensing Utilizing Mass Amplification and Magnetic Bead Convection

    Journal: Micromachines

    doi: 10.3390/mi9040194

    Close-up of the fs-laser fabricated pole pieces along the microfluidic channel with immobilized superparamagnetic beads.
    Figure Legend Snippet: Close-up of the fs-laser fabricated pole pieces along the microfluidic channel with immobilized superparamagnetic beads.

    Techniques Used:

    C-reactive protein (CRP) measurement utilizing the mass enhancement protocol involving a second antibody and streptavidin-coated superparamagnetic nanoparticles.
    Figure Legend Snippet: C-reactive protein (CRP) measurement utilizing the mass enhancement protocol involving a second antibody and streptavidin-coated superparamagnetic nanoparticles.

    Techniques Used:

    37) Product Images from "A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)"

    Article Title: A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)

    Journal: Journal of Virology

    doi: 10.1128/JVI.00763-17

    Biochemical validation of interactions between arenavirus NPs expressed from plasmids and endogenous host proteins. (A) HEK 293T cells were cotransfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide, along with a second plasmid that encodes BirA, a bacterial biotin ligase, to ensure biotinylation of the viral NPs. As a control, cells were cotransfected with the BirA plasmid and an empty vector (p0). Biotinylated NPs and associated host proteins were affinity purified from cell lysates (input) by use of magnetic streptavidin beads, and captured proteins were detected by Western blotting. (B and C) HEK 293T cells were transfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide or, as a control, an empty vector (p0). (B) PKR (bait) was immunoprecipitated with a monoclonal antibody to the C terminus of PKR or with an irrelevant rabbit IgG. PKR (bait) and the associated viral NP (prey) were detected by Western blotting. (C) AIFM1 (bait) was immunoprecipitated with a polyclonal antibody. AIFM1 (bait) and the associated viral NP (prey) were detected by Western blotting. Data are representative of 2 (A), 3 (B), or 2 (C) independent experiments.
    Figure Legend Snippet: Biochemical validation of interactions between arenavirus NPs expressed from plasmids and endogenous host proteins. (A) HEK 293T cells were cotransfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide, along with a second plasmid that encodes BirA, a bacterial biotin ligase, to ensure biotinylation of the viral NPs. As a control, cells were cotransfected with the BirA plasmid and an empty vector (p0). Biotinylated NPs and associated host proteins were affinity purified from cell lysates (input) by use of magnetic streptavidin beads, and captured proteins were detected by Western blotting. (B and C) HEK 293T cells were transfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide or, as a control, an empty vector (p0). (B) PKR (bait) was immunoprecipitated with a monoclonal antibody to the C terminus of PKR or with an irrelevant rabbit IgG. PKR (bait) and the associated viral NP (prey) were detected by Western blotting. (C) AIFM1 (bait) was immunoprecipitated with a polyclonal antibody. AIFM1 (bait) and the associated viral NP (prey) were detected by Western blotting. Data are representative of 2 (A), 3 (B), or 2 (C) independent experiments.

    Techniques Used: Plasmid Preparation, Affinity Purification, Western Blot, Transfection, Immunoprecipitation

    38) Product Images from "NEDD4 family ubiquitin ligases associate with LCMV Z’s PPXY domain and are required for virus budding, but not via direct ubiquitination of Z"

    Article Title: NEDD4 family ubiquitin ligases associate with LCMV Z’s PPXY domain and are required for virus budding, but not via direct ubiquitination of Z

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008100

    Identification of host proteins that interact with the LCMV and LASV Z matrix proteins or are packaged into LCMV virions. (A) The flow diagram depicts the affinity purification- and mass spectrometry-based approach used to identify human protein partners of LCMV or LASV Z proteins. HEK293T cells were transfected with a plasmid encoding LCMV or LASV Z with a C-terminal streptavidin binding peptide (SBP) tag or an empty vector. Z and its human protein binding partners were affinity purified from cell lysates or cell-free virus-like particles (VLPs) using magnetic streptavidin beads and the captured protein complexes were separated by protein gel electrophoresis and processed for mass spectrometry as described in the Methods. The Coomassie-stained protein gel shown is representative of two independent experiments. (B) The flow diagram shows the sucrose banding- and mass spectrometry-based approach to detect host proteins in LCMV particles. Intact virions of LCMV strain Armstrong 53b produced in Vero E6 cells were sucrose-banded and then subjected to lysis followed by protein gel electrophoresis. The Coomassie stained gel, which was processed for mass spectrometry as described in the Methods, is shown and various viral proteins are indicated.
    Figure Legend Snippet: Identification of host proteins that interact with the LCMV and LASV Z matrix proteins or are packaged into LCMV virions. (A) The flow diagram depicts the affinity purification- and mass spectrometry-based approach used to identify human protein partners of LCMV or LASV Z proteins. HEK293T cells were transfected with a plasmid encoding LCMV or LASV Z with a C-terminal streptavidin binding peptide (SBP) tag or an empty vector. Z and its human protein binding partners were affinity purified from cell lysates or cell-free virus-like particles (VLPs) using magnetic streptavidin beads and the captured protein complexes were separated by protein gel electrophoresis and processed for mass spectrometry as described in the Methods. The Coomassie-stained protein gel shown is representative of two independent experiments. (B) The flow diagram shows the sucrose banding- and mass spectrometry-based approach to detect host proteins in LCMV particles. Intact virions of LCMV strain Armstrong 53b produced in Vero E6 cells were sucrose-banded and then subjected to lysis followed by protein gel electrophoresis. The Coomassie stained gel, which was processed for mass spectrometry as described in the Methods, is shown and various viral proteins are indicated.

    Techniques Used: Flow Cytometry, Affinity Purification, Mass Spectrometry, Transfection, Plasmid Preparation, Binding Assay, Protein Binding, Nucleic Acid Electrophoresis, Staining, Produced, Lysis

    The LCMV Z protein is directly ubiquitinated on lysine residues by Nedd4 family ubiquitin ligases. (A) SBP-tagged LCMV Z (WT or Z with the tyrosine (88) in the PPXY late domain mutated), Junín virus (JUNV) Z, or an empty vector were expressed via plasmid in HEK293T cells. Z was affinity purified with streptavidin-coated magnetic beads and Z along with the Nedd4 family ubiquitin ligases ITCH, WWP1, and Nedd4 were detected via western blot. (B) SBP-tagged LCMV Z and hemagglutinin (HA)-tagged ubiquitin were expressed via plasmid in HEK293T cells and two days later Z was affinity purified (AP) with streptavidin beads and both Z and ubiquitin (Ub) were detected via two color fluorescent western blotting using anti-SBP and anti-HA antibodies. The Z monomeric band is indicated as well as overlapping bands at ~32kDa which may represent an ubiquitinated Z species (Z-Ub). (C) To distinguish between ubiquitinated host proteins and ubiquitinated Z, plasmids expressing either WT or lysine-free (NoK) LCMV Z with affinity tags of different molecular weights (SBP or Strep tag II) were co-transfected with HA-tagged ubiquitin in HEK293T cells and two days later Z was affinity purified with streptavidin beads and both Z and ubiquitin were detected via two color fluorescent western blotting using a combination of anti-SBP, anti-Strep tag II, and anti-HA antibodies. (D) In order to eliminate non-covalently interacting host proteins from co-purifying with LCMV Z, urea was added to the cell lysate to a concentration of 8M and incubated for 1 hour. The samples were then diluted with lysis buffer to reduce the urea concentration to 2 M and a streptavidin purification was performed. Western blotting was used to detect Z and ubiquitin as well as the known LCMV Z host partners IMPDH2 and ATP5B. (E) HEK293T cells were seeded then one day later small interfering (si)RNAs targeting ITCH, Nedd4, and WWP1 or a concentration of a non-targeting siRNA equal to the total of the three distinct siRNAs were transfected into the cells and two days later the cells were transfected with plasmids expressing SBP-tagged LCMV Z and HA-tagged ubiquitin. One day after the plasmid transfection, the cells were lysed and Z was affinity purified with streptavidin beads and was detected along with ubiquitin by two color fluorescent western blotting. (F-G) HEK293T cells were transfected with plasmids expressing LCMV Z WT or lysine-free Z (NoK) and HA-ubiquitin and treated with the indicated concentrations of the PPXY-Nedd4 interaction inhibitor, compound #4. Two days later the cells were collected and Z was purified with streptavidin beads from cell lysates and ubiquitin and Z were detected by two color fluorescent western blotting. (G) The quantity of the Z-Ub bands in the fluorescent western blots in (F) were quantified using Licor Image Studio software and divided by the quantity of monomeric Z from three independent experiments for all conditions except the 6μM and NoK DMSO conditions which were included only twice. Data in (F) represent the mean ± SEM of these experiments and a one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the WT DMSO condition. **p
    Figure Legend Snippet: The LCMV Z protein is directly ubiquitinated on lysine residues by Nedd4 family ubiquitin ligases. (A) SBP-tagged LCMV Z (WT or Z with the tyrosine (88) in the PPXY late domain mutated), Junín virus (JUNV) Z, or an empty vector were expressed via plasmid in HEK293T cells. Z was affinity purified with streptavidin-coated magnetic beads and Z along with the Nedd4 family ubiquitin ligases ITCH, WWP1, and Nedd4 were detected via western blot. (B) SBP-tagged LCMV Z and hemagglutinin (HA)-tagged ubiquitin were expressed via plasmid in HEK293T cells and two days later Z was affinity purified (AP) with streptavidin beads and both Z and ubiquitin (Ub) were detected via two color fluorescent western blotting using anti-SBP and anti-HA antibodies. The Z monomeric band is indicated as well as overlapping bands at ~32kDa which may represent an ubiquitinated Z species (Z-Ub). (C) To distinguish between ubiquitinated host proteins and ubiquitinated Z, plasmids expressing either WT or lysine-free (NoK) LCMV Z with affinity tags of different molecular weights (SBP or Strep tag II) were co-transfected with HA-tagged ubiquitin in HEK293T cells and two days later Z was affinity purified with streptavidin beads and both Z and ubiquitin were detected via two color fluorescent western blotting using a combination of anti-SBP, anti-Strep tag II, and anti-HA antibodies. (D) In order to eliminate non-covalently interacting host proteins from co-purifying with LCMV Z, urea was added to the cell lysate to a concentration of 8M and incubated for 1 hour. The samples were then diluted with lysis buffer to reduce the urea concentration to 2 M and a streptavidin purification was performed. Western blotting was used to detect Z and ubiquitin as well as the known LCMV Z host partners IMPDH2 and ATP5B. (E) HEK293T cells were seeded then one day later small interfering (si)RNAs targeting ITCH, Nedd4, and WWP1 or a concentration of a non-targeting siRNA equal to the total of the three distinct siRNAs were transfected into the cells and two days later the cells were transfected with plasmids expressing SBP-tagged LCMV Z and HA-tagged ubiquitin. One day after the plasmid transfection, the cells were lysed and Z was affinity purified with streptavidin beads and was detected along with ubiquitin by two color fluorescent western blotting. (F-G) HEK293T cells were transfected with plasmids expressing LCMV Z WT or lysine-free Z (NoK) and HA-ubiquitin and treated with the indicated concentrations of the PPXY-Nedd4 interaction inhibitor, compound #4. Two days later the cells were collected and Z was purified with streptavidin beads from cell lysates and ubiquitin and Z were detected by two color fluorescent western blotting. (G) The quantity of the Z-Ub bands in the fluorescent western blots in (F) were quantified using Licor Image Studio software and divided by the quantity of monomeric Z from three independent experiments for all conditions except the 6μM and NoK DMSO conditions which were included only twice. Data in (F) represent the mean ± SEM of these experiments and a one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the WT DMSO condition. **p

    Techniques Used: Plasmid Preparation, Affinity Purification, Magnetic Beads, Western Blot, Expressing, Strep-tag, Transfection, Concentration Assay, Incubation, Lysis, Purification, Software

    Efficient interaction of LCMV Z with the ESCRT protein VPS4 requires residues K10, K77, and the PPXY late domain. (A-B) HEK293T cells were transfected with an empty vector or a vector encoding SBP-tagged LCMV Z with the indicated mutations. Z was affinity purified using streptavidin beads and the purified Z (bait) and VPS4 (prey) were detected by western blotting. (B) The quantity of co-purified VPS4 in the fluorescent western blots in (A) was quantitated using Licor Image Studio software and divided by the quantity of affinity purified monomeric Z. (C-D) HEK293T cells were transfected with plasmids expressing HA-tagged ubiquitin and either an empty vector or a vector encoding SBP-tagged LCMV Z with the indicated mutations. Z was affinity purified using streptavidin beads and the purified Z (bait) and VPS4 (prey) were detected by western blotting as described in (A). (D) The quantity of co-purified VPS4 in the fluorescent western blots in (C) was quantified using Licor Image Studio software and divided by the quantity of affinity purified monomeric Z. (E) HEK293T cells were transfected with plasmids expressing HA-tagged ubiquitin and SBP-tagged WT or NoK LCMV Z and treated with the indicated doses of compound #4 or DMSO vehicle control. Z was affinity purified after two days and Z and VPS4 were detected by western blotting. The western blots shown are representative of three independent experiments. The graphs in (B) and (D) represent the mean values ± SEM of three independent experiments. A one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the WT control in (B) and (D). *p
    Figure Legend Snippet: Efficient interaction of LCMV Z with the ESCRT protein VPS4 requires residues K10, K77, and the PPXY late domain. (A-B) HEK293T cells were transfected with an empty vector or a vector encoding SBP-tagged LCMV Z with the indicated mutations. Z was affinity purified using streptavidin beads and the purified Z (bait) and VPS4 (prey) were detected by western blotting. (B) The quantity of co-purified VPS4 in the fluorescent western blots in (A) was quantitated using Licor Image Studio software and divided by the quantity of affinity purified monomeric Z. (C-D) HEK293T cells were transfected with plasmids expressing HA-tagged ubiquitin and either an empty vector or a vector encoding SBP-tagged LCMV Z with the indicated mutations. Z was affinity purified using streptavidin beads and the purified Z (bait) and VPS4 (prey) were detected by western blotting as described in (A). (D) The quantity of co-purified VPS4 in the fluorescent western blots in (C) was quantified using Licor Image Studio software and divided by the quantity of affinity purified monomeric Z. (E) HEK293T cells were transfected with plasmids expressing HA-tagged ubiquitin and SBP-tagged WT or NoK LCMV Z and treated with the indicated doses of compound #4 or DMSO vehicle control. Z was affinity purified after two days and Z and VPS4 were detected by western blotting. The western blots shown are representative of three independent experiments. The graphs in (B) and (D) represent the mean values ± SEM of three independent experiments. A one-way ANOVA with Holm-Sidak’s test for multiple comparisons was used to compare the mean values to the WT control in (B) and (D). *p

    Techniques Used: Transfection, Plasmid Preparation, Affinity Purification, Purification, Western Blot, Software, Expressing

    39) Product Images from "PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells"

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

    Journal: eLife

    doi: 10.7554/eLife.14470

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    40) Product Images from "Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs"

    Article Title: Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0120043

    Affinity purification of cDNAs primed with biotinylated RT primers circumvents nonspecific priming during RT. (A and B) RNA extracted from a high titer stock of cell-free LCMV virions was subjected to standard RT-PCR to detect S segment vRNA using the RT primer S 2865- and PCR primers 1856+ and 2628- (note that the sequence for primer 1856+ is listed in the Methods). In panel (A), three RT conditions were tested. The first RT condition featured a standard RT primer, the second had a biotinylated primer, and the third had no RT primer, as indicated. A portion of each reaction was subjected to affinity purification using streptavidin magnetic beads and then both the input and streptavidin-purified cDNAs were subjected to PCR. In panel (B), two RT conditions were tested: one with a biotinylated RT primer and the other without an RT primer. In an attempt to eliminate nonbiotinylated cDNAs from nonspecifically binding to streptavidin beads, a panel of four wash buffers (the 2X wash buffer from the Dynabeads kilobaseBINDER Kit, a 1X dilution of this buffer alone or containing 0.5% Tween 20, or water containing 0.5% Tween 20) were used during affinity purification. Following affinity purification, the captured cDNAs were subjected to PCR.
    Figure Legend Snippet: Affinity purification of cDNAs primed with biotinylated RT primers circumvents nonspecific priming during RT. (A and B) RNA extracted from a high titer stock of cell-free LCMV virions was subjected to standard RT-PCR to detect S segment vRNA using the RT primer S 2865- and PCR primers 1856+ and 2628- (note that the sequence for primer 1856+ is listed in the Methods). In panel (A), three RT conditions were tested. The first RT condition featured a standard RT primer, the second had a biotinylated primer, and the third had no RT primer, as indicated. A portion of each reaction was subjected to affinity purification using streptavidin magnetic beads and then both the input and streptavidin-purified cDNAs were subjected to PCR. In panel (B), two RT conditions were tested: one with a biotinylated RT primer and the other without an RT primer. In an attempt to eliminate nonbiotinylated cDNAs from nonspecifically binding to streptavidin beads, a panel of four wash buffers (the 2X wash buffer from the Dynabeads kilobaseBINDER Kit, a 1X dilution of this buffer alone or containing 0.5% Tween 20, or water containing 0.5% Tween 20) were used during affinity purification. Following affinity purification, the captured cDNAs were subjected to PCR.

    Techniques Used: Affinity Purification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Magnetic Beads, Purification, Binding Assay

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    Article Snippet: .. Finally, cells were stained with anti-MCL1 (#5453) and TOPRO3 (Invitrogen) for 1 hour at room temperature and then imaged using the Zeiss LSM510 Meta confocal microscope. .. Caspase-Glo 3/7 assay was performed according to manufacturer’s instruction (Promega).

    Incubation:

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    Microscopy:

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    Blocking Assay:

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    Proteomic screen identifies ESCRT components as EphB2 interactors. (A) Strategy of purification and identification of the interactome of biotinylated EphB2 by mass spectrometry. (B) Representative images showing clustering of biotinylated FLAG-Avi-EphB2-YFP fusion protein around <t>streptavidin-conjugated</t> <t>Dynabeads</t> (within 5 min, right, stippled line). This effect required EphB2 biotinylation (left). (C) Western blot analysis showing tyrosine autophosphorylation (detected by anti-phospho EphB2 [Y594] or 4G10 antibodies) of biotinylated (BirA + ) but not unbiotinylated (BirA – ) FLAG-Avi-EphB2 in response to incubation with streptavidin-conjugated Dynabeads. Unfused Fc protein was used as negative control. Similar results were observed in three independent replicates. (D) Bar graph showing the SILAC ratios of representative members of different groups of the top 30 enriched full-length EphB2 interactors ( n = 3, mean ± SEM). Yellow, biotinylated versus unbiotinylated full-length EphB2 (FL EphB2); blue, biotinylated EphB2-ΔC versus unbiotinylated full-length EphB2 (EphB2-ΔC); gray, biotinylated full-length EphB2 versus biotinylated EphB2-ΔC (EphB2-cyto). (E) Full list of ESCRT complex components identified in the proteomic screen as interactors of full-length EphB2 in at least one of the three replicates. (F) Validation of the interaction between EphB2 and endogenous STAM1 or VPS4A by co-IP/Western blot (WB) analysis in HEK293 cells stably expressing EphB2. (G) Validation of the interaction between overexpressed EphB2 and STAM1 in HeLa cells. (H) Representative images showing that endogenous STAM and CHMP4B colocalize with surface EphB2 in HeLa cells. STAM and CHMP4B levels at the plasma membrane were increased 3.5- and 2-fold, respectively (highlighted by red triangles), in EphB2 + cells (indicated by yellow stippled line in the merge) compared with untransfected cells (white stippled line). Bars, 10 µm.
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    Thermo Fisher streptavidin coated beads
    Amyloid peptide 12B interacts with influenza via the PB2 target protein in cellulo. a Quantification of the area covered by plaques of A/PR8-infected MDCK cells treated with 10 µM peptide 12B at different time points relative to infection. Data are normalized to medium-treated cells and mean ± SD is shown ( n = 8 from three independent experiments, statistics: one-way ANOVA with multiple comparison, p value = 0.0605). Representative images are shown in Supplementary Fig. 5a . b Quantification of the area covered by plaques in a plaque-size reduction assay of MDCK cells infected with A/PR8 virion particles that were pretreated with peptide 12B. Concentration-dependent effect of peptide 12B is shown normalized to medium-treated virion particles as mean ± SD ( n = 3 independent experiments). Representative images are shown in Supplementary Fig. 5b . c MDCK cells treated with FITC-labeled peptide 12B, 12Bpro2 (10 µM), or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Cells were fixed and stained with an anti-FLAG antibody and DAPI. One representative example of three independent experiments. d Western blot analysis of total (input) and immunoprecipitated fraction of MDCK cells treated with PEG 2 -biotin-labeled peptide (10 µM) or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Quantification of immunoprecipitated fraction of PB2, relative to PB2 input levels, is shown on the right. Data represent mean ± SD ( n = 5 independent experiments, statistics: one-way ANOVA with multiple comparison). <t>Streptavidin</t> was used as a loading control. e Western blot analysis of PB2 distribution in soluble and insoluble fraction of lysates of MDCK cells treated with peptide (10 µM) for 2 h prior to influenza A/WSN-FLAG infection (MOI = 1, 16-h infection). Vimentin and GAPDH were used as loading controls for insoluble and soluble fraction, respectively. Quantification represents soluble PB2 fraction, normalized to medium-treated cells (100% soluble PB2). Data represent mean ± SD ( n = 4 independent experiments, statistics: one-way ANOVA with multiple comparison). For d and e the samples on one blot are derived from the same experiment and the gels/blots were processed in parallel. Full blots are shown in Supplementary Fig. 12 .
    Streptavidin Coated Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher t7 separate library streptavidin coupled magnetic beads
    SEPARATE design and workflow. (A) An oligonucleotide library encoding > 29,000 human protein isoforms as 45 amino acid overlapping 90-mer tiles is synthesized on a DNA microarray and cloned into the T7-SEPARTATE vector. This monovalent display vector flanks a library peptide by an N-terminal 3xFLAG tag and a C-terminal biotin labeling tag. (B) The biotin tagged library is immobilized on streptavidin-coupled magnetic beads, digested with a protease containing solution and digested phage clones are recaptured on M2 FLAG antibody coated protein G beads. (C) Recaptured phage clones are amplified by PCR and their clonal abundance quantified by deep sequencing to generate a fold-enrichment matrix. (D) <t>T7-SEPARATE</t> clones are biotin labeled in vivo and can be immobilized on streptavidin-coupled magnetic beads. (E) A T7-SEPARATE clone encoding the PreScission cleavage motif -LGVLPG/GP- was prepared using both the T7Select1-2b monovalent (○) and the T7Select1-3b multivalent (◇) T7 vector scaffolds. A negative control clone lacking the PreScission cleavage motif is not enriched on M2 FLAG antibody coated beads for both monovalent (●) and multivalent (◆) display scaffolds. (F) The PreScission digestible clone spiked into the human library demonstrates a dose-dependent enrichment (●); no enrichment is observed when digested with Caspase-1 (○). (G) MA plot analysis from a PreScission digest compares a peptide’s enrichment (Log[Protease/Buffer]) against its relative abundance in the human library (0.5*Log[Protease*Buffer]).
    T7 Separate Library Streptavidin Coupled Magnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Proteomic screen identifies ESCRT components as EphB2 interactors. (A) Strategy of purification and identification of the interactome of biotinylated EphB2 by mass spectrometry. (B) Representative images showing clustering of biotinylated FLAG-Avi-EphB2-YFP fusion protein around streptavidin-conjugated Dynabeads (within 5 min, right, stippled line). This effect required EphB2 biotinylation (left). (C) Western blot analysis showing tyrosine autophosphorylation (detected by anti-phospho EphB2 [Y594] or 4G10 antibodies) of biotinylated (BirA + ) but not unbiotinylated (BirA – ) FLAG-Avi-EphB2 in response to incubation with streptavidin-conjugated Dynabeads. Unfused Fc protein was used as negative control. Similar results were observed in three independent replicates. (D) Bar graph showing the SILAC ratios of representative members of different groups of the top 30 enriched full-length EphB2 interactors ( n = 3, mean ± SEM). Yellow, biotinylated versus unbiotinylated full-length EphB2 (FL EphB2); blue, biotinylated EphB2-ΔC versus unbiotinylated full-length EphB2 (EphB2-ΔC); gray, biotinylated full-length EphB2 versus biotinylated EphB2-ΔC (EphB2-cyto). (E) Full list of ESCRT complex components identified in the proteomic screen as interactors of full-length EphB2 in at least one of the three replicates. (F) Validation of the interaction between EphB2 and endogenous STAM1 or VPS4A by co-IP/Western blot (WB) analysis in HEK293 cells stably expressing EphB2. (G) Validation of the interaction between overexpressed EphB2 and STAM1 in HeLa cells. (H) Representative images showing that endogenous STAM and CHMP4B colocalize with surface EphB2 in HeLa cells. STAM and CHMP4B levels at the plasma membrane were increased 3.5- and 2-fold, respectively (highlighted by red triangles), in EphB2 + cells (indicated by yellow stippled line in the merge) compared with untransfected cells (white stippled line). Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Exosomes mediate cell contact–independent ephrin-Eph signaling during axon guidance

    doi: 10.1083/jcb.201601085

    Figure Lengend Snippet: Proteomic screen identifies ESCRT components as EphB2 interactors. (A) Strategy of purification and identification of the interactome of biotinylated EphB2 by mass spectrometry. (B) Representative images showing clustering of biotinylated FLAG-Avi-EphB2-YFP fusion protein around streptavidin-conjugated Dynabeads (within 5 min, right, stippled line). This effect required EphB2 biotinylation (left). (C) Western blot analysis showing tyrosine autophosphorylation (detected by anti-phospho EphB2 [Y594] or 4G10 antibodies) of biotinylated (BirA + ) but not unbiotinylated (BirA – ) FLAG-Avi-EphB2 in response to incubation with streptavidin-conjugated Dynabeads. Unfused Fc protein was used as negative control. Similar results were observed in three independent replicates. (D) Bar graph showing the SILAC ratios of representative members of different groups of the top 30 enriched full-length EphB2 interactors ( n = 3, mean ± SEM). Yellow, biotinylated versus unbiotinylated full-length EphB2 (FL EphB2); blue, biotinylated EphB2-ΔC versus unbiotinylated full-length EphB2 (EphB2-ΔC); gray, biotinylated full-length EphB2 versus biotinylated EphB2-ΔC (EphB2-cyto). (E) Full list of ESCRT complex components identified in the proteomic screen as interactors of full-length EphB2 in at least one of the three replicates. (F) Validation of the interaction between EphB2 and endogenous STAM1 or VPS4A by co-IP/Western blot (WB) analysis in HEK293 cells stably expressing EphB2. (G) Validation of the interaction between overexpressed EphB2 and STAM1 in HeLa cells. (H) Representative images showing that endogenous STAM and CHMP4B colocalize with surface EphB2 in HeLa cells. STAM and CHMP4B levels at the plasma membrane were increased 3.5- and 2-fold, respectively (highlighted by red triangles), in EphB2 + cells (indicated by yellow stippled line in the merge) compared with untransfected cells (white stippled line). Bars, 10 µm.

    Article Snippet: 30 µl of streptavidin-conjugated Dynabeads (Dynabeads M-280 Streptavidin; Thermo Fisher Scientific) were added per dish to induce EphB2 clustering.

    Techniques: Purification, Mass Spectrometry, Western Blot, Incubation, Negative Control, Co-Immunoprecipitation Assay, Stable Transfection, Expressing

    Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated

    Journal: Blood

    Article Title: Biochemical and structural analysis of the interaction between β-amyloid and fibrinogen

    doi: 10.1182/blood-2016-03-705228

    Figure Lengend Snippet: Aβ22-41 binds to fibrinogen and fragment D. (A-B) Biotin-labeled Aβ42, Aβ1-16, Aβ15-25, and Aβ22-41 were incubated with fibrinogen (FBG) or fragment D (FD), and pulldown assays were carried out using streptavidin-coated

    Article Snippet: Streptavidin-coated magnetic beads (Dynabeads M-280; Thermo-Fisher) were added for 30 minutes, washed, and eluted with nonreducing 1× lithium dodecyl sulfate sample buffer (Thermo Fisher Scientific).

    Techniques: Labeling, Incubation

    Amyloid peptide 12B interacts with influenza via the PB2 target protein in cellulo. a Quantification of the area covered by plaques of A/PR8-infected MDCK cells treated with 10 µM peptide 12B at different time points relative to infection. Data are normalized to medium-treated cells and mean ± SD is shown ( n = 8 from three independent experiments, statistics: one-way ANOVA with multiple comparison, p value = 0.0605). Representative images are shown in Supplementary Fig. 5a . b Quantification of the area covered by plaques in a plaque-size reduction assay of MDCK cells infected with A/PR8 virion particles that were pretreated with peptide 12B. Concentration-dependent effect of peptide 12B is shown normalized to medium-treated virion particles as mean ± SD ( n = 3 independent experiments). Representative images are shown in Supplementary Fig. 5b . c MDCK cells treated with FITC-labeled peptide 12B, 12Bpro2 (10 µM), or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Cells were fixed and stained with an anti-FLAG antibody and DAPI. One representative example of three independent experiments. d Western blot analysis of total (input) and immunoprecipitated fraction of MDCK cells treated with PEG 2 -biotin-labeled peptide (10 µM) or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Quantification of immunoprecipitated fraction of PB2, relative to PB2 input levels, is shown on the right. Data represent mean ± SD ( n = 5 independent experiments, statistics: one-way ANOVA with multiple comparison). Streptavidin was used as a loading control. e Western blot analysis of PB2 distribution in soluble and insoluble fraction of lysates of MDCK cells treated with peptide (10 µM) for 2 h prior to influenza A/WSN-FLAG infection (MOI = 1, 16-h infection). Vimentin and GAPDH were used as loading controls for insoluble and soluble fraction, respectively. Quantification represents soluble PB2 fraction, normalized to medium-treated cells (100% soluble PB2). Data represent mean ± SD ( n = 4 independent experiments, statistics: one-way ANOVA with multiple comparison). For d and e the samples on one blot are derived from the same experiment and the gels/blots were processed in parallel. Full blots are shown in Supplementary Fig. 12 .

    Journal: Nature Communications

    Article Title: Reverse engineering synthetic antiviral amyloids

    doi: 10.1038/s41467-020-16721-8

    Figure Lengend Snippet: Amyloid peptide 12B interacts with influenza via the PB2 target protein in cellulo. a Quantification of the area covered by plaques of A/PR8-infected MDCK cells treated with 10 µM peptide 12B at different time points relative to infection. Data are normalized to medium-treated cells and mean ± SD is shown ( n = 8 from three independent experiments, statistics: one-way ANOVA with multiple comparison, p value = 0.0605). Representative images are shown in Supplementary Fig. 5a . b Quantification of the area covered by plaques in a plaque-size reduction assay of MDCK cells infected with A/PR8 virion particles that were pretreated with peptide 12B. Concentration-dependent effect of peptide 12B is shown normalized to medium-treated virion particles as mean ± SD ( n = 3 independent experiments). Representative images are shown in Supplementary Fig. 5b . c MDCK cells treated with FITC-labeled peptide 12B, 12Bpro2 (10 µM), or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Cells were fixed and stained with an anti-FLAG antibody and DAPI. One representative example of three independent experiments. d Western blot analysis of total (input) and immunoprecipitated fraction of MDCK cells treated with PEG 2 -biotin-labeled peptide (10 µM) or buffer for 2 h prior to A/WSN-FLAG infection (MOI = 1, 16-h infection). Quantification of immunoprecipitated fraction of PB2, relative to PB2 input levels, is shown on the right. Data represent mean ± SD ( n = 5 independent experiments, statistics: one-way ANOVA with multiple comparison). Streptavidin was used as a loading control. e Western blot analysis of PB2 distribution in soluble and insoluble fraction of lysates of MDCK cells treated with peptide (10 µM) for 2 h prior to influenza A/WSN-FLAG infection (MOI = 1, 16-h infection). Vimentin and GAPDH were used as loading controls for insoluble and soluble fraction, respectively. Quantification represents soluble PB2 fraction, normalized to medium-treated cells (100% soluble PB2). Data represent mean ± SD ( n = 4 independent experiments, statistics: one-way ANOVA with multiple comparison). For d and e the samples on one blot are derived from the same experiment and the gels/blots were processed in parallel. Full blots are shown in Supplementary Fig. 12 .

    Article Snippet: Lysates were then mixed with streptavidin-coated beads (Dynabeads™ M-280 Streptavidin, Thermo Fisher Scientific) and rotated for 1 h at 4 °C.

    Techniques: Infection, Concentration Assay, Labeling, Staining, Western Blot, Immunoprecipitation, Derivative Assay

    SEPARATE design and workflow. (A) An oligonucleotide library encoding > 29,000 human protein isoforms as 45 amino acid overlapping 90-mer tiles is synthesized on a DNA microarray and cloned into the T7-SEPARTATE vector. This monovalent display vector flanks a library peptide by an N-terminal 3xFLAG tag and a C-terminal biotin labeling tag. (B) The biotin tagged library is immobilized on streptavidin-coupled magnetic beads, digested with a protease containing solution and digested phage clones are recaptured on M2 FLAG antibody coated protein G beads. (C) Recaptured phage clones are amplified by PCR and their clonal abundance quantified by deep sequencing to generate a fold-enrichment matrix. (D) T7-SEPARATE clones are biotin labeled in vivo and can be immobilized on streptavidin-coupled magnetic beads. (E) A T7-SEPARATE clone encoding the PreScission cleavage motif -LGVLPG/GP- was prepared using both the T7Select1-2b monovalent (○) and the T7Select1-3b multivalent (◇) T7 vector scaffolds. A negative control clone lacking the PreScission cleavage motif is not enriched on M2 FLAG antibody coated beads for both monovalent (●) and multivalent (◆) display scaffolds. (F) The PreScission digestible clone spiked into the human library demonstrates a dose-dependent enrichment (●); no enrichment is observed when digested with Caspase-1 (○). (G) MA plot analysis from a PreScission digest compares a peptide’s enrichment (Log[Protease/Buffer]) against its relative abundance in the human library (0.5*Log[Protease*Buffer]).

    Journal: bioRxiv

    Article Title: Protease Activity Profiling Via Programmable Phage Display

    doi: 10.1101/2020.05.11.089607

    Figure Lengend Snippet: SEPARATE design and workflow. (A) An oligonucleotide library encoding > 29,000 human protein isoforms as 45 amino acid overlapping 90-mer tiles is synthesized on a DNA microarray and cloned into the T7-SEPARTATE vector. This monovalent display vector flanks a library peptide by an N-terminal 3xFLAG tag and a C-terminal biotin labeling tag. (B) The biotin tagged library is immobilized on streptavidin-coupled magnetic beads, digested with a protease containing solution and digested phage clones are recaptured on M2 FLAG antibody coated protein G beads. (C) Recaptured phage clones are amplified by PCR and their clonal abundance quantified by deep sequencing to generate a fold-enrichment matrix. (D) T7-SEPARATE clones are biotin labeled in vivo and can be immobilized on streptavidin-coupled magnetic beads. (E) A T7-SEPARATE clone encoding the PreScission cleavage motif -LGVLPG/GP- was prepared using both the T7Select1-2b monovalent (○) and the T7Select1-3b multivalent (◇) T7 vector scaffolds. A negative control clone lacking the PreScission cleavage motif is not enriched on M2 FLAG antibody coated beads for both monovalent (●) and multivalent (◆) display scaffolds. (F) The PreScission digestible clone spiked into the human library demonstrates a dose-dependent enrichment (●); no enrichment is observed when digested with Caspase-1 (○). (G) MA plot analysis from a PreScission digest compares a peptide’s enrichment (Log[Protease/Buffer]) against its relative abundance in the human library (0.5*Log[Protease*Buffer]).

    Article Snippet: SEPARATE Assay: Immobilization of the T7-SEPARATE Library Streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, ThermoFisher Scientific) were washed (TBS, pH 7.4, 0.01% NP40) and resuspended in binding buffer (TBS, pH 7.4, 0.001% NP40) containing 2E9 plaque forming units per 10 μl of bead slurry.

    Techniques: Synthesized, Microarray, Clone Assay, Plasmid Preparation, Labeling, Magnetic Beads, Amplification, Polymerase Chain Reaction, Sequencing, In Vivo, Negative Control

    A T7-SEPARATE clone encoding the PreScission cleavage motif -LGVLPG/GP- was prepared using both the T7Select1-2b monovalent (○) and the T7Select10-3b multivalent (◇) T7 display vectors. The monovalent display results in a higher depletion of phage clones from the immobilization beads indicating a higher fraction is digested at a given concentration of PreScission protease. A negative control clone lacking the PreScission cleavage motif is not digested off of streptavidin-coated beads for both monovalent (●) and multivalent (◆) display vectors.

    Journal: bioRxiv

    Article Title: Protease Activity Profiling Via Programmable Phage Display

    doi: 10.1101/2020.05.11.089607

    Figure Lengend Snippet: A T7-SEPARATE clone encoding the PreScission cleavage motif -LGVLPG/GP- was prepared using both the T7Select1-2b monovalent (○) and the T7Select10-3b multivalent (◇) T7 display vectors. The monovalent display results in a higher depletion of phage clones from the immobilization beads indicating a higher fraction is digested at a given concentration of PreScission protease. A negative control clone lacking the PreScission cleavage motif is not digested off of streptavidin-coated beads for both monovalent (●) and multivalent (◆) display vectors.

    Article Snippet: SEPARATE Assay: Immobilization of the T7-SEPARATE Library Streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, ThermoFisher Scientific) were washed (TBS, pH 7.4, 0.01% NP40) and resuspended in binding buffer (TBS, pH 7.4, 0.001% NP40) containing 2E9 plaque forming units per 10 μl of bead slurry.

    Techniques: Clone Assay, Concentration Assay, Negative Control