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  • 99
    Thermo Fisher superase in rnase inhibitor
    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with <t>SUPERase-In™</t> <t>RNase</t> Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.
    Superase In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher superase in
    SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor <t>Superase-In</t> (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.
    Superase In, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2546 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1031 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Fisher Scientific superase in
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase In, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher superase intm
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase Intm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher superase ln
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase Ln, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher superase ∙
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase ∙, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega superase
    Trans-protection of mRNA against skin surface ribonuclease activity . Ribonuclease activity assay (negative images). Capped lacZ mRNA in the absence (w/o) or presence of the ribonuclease inhibitors RNasin ® (R-in, final concentration 1 U/μl) or <t>SUPERase·</t> In™ (S-in, 1 U/μl) was incubated with ribonucleases of Homo sapiens hand surface (final dilution 4×) or of Mus musculus ear surface (20×) or with 2, 5 pg/μl ribonuclease A from Bos taurus pancreas at 37°C for increasing time (indicated by the wedge): Samples were taken immediately after addition of ribonucleases as well as 15 min and 60 min after. Samples without ribonucleases are indicated by a dash and were not incubated at 37°C.
    Superase, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Journal: Cell Cycle

    Article Title: FEM1 proteins are ancient regulators of SLBP degradation

    doi: 10.1080/15384101.2017.1284715

    Figure Lengend Snippet: Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Article Snippet: SUPERase-In™ RNase Inhibitor (Thermo Fisher Scientific) was used at 1U/μL where indicated.

    Techniques: Binding Assay, Sequencing, Activation Assay, RNA Binding Assay, Transfection, Plasmid Preparation, Construct, Mutagenesis, Immunoprecipitation

    Whole saliva was left untreated or stabilized with RNAlater, Superase or RPS. RNA was extracted immediately or after incubation at room temperature for one week. The transcriptomes of fresh and stored aliquots for each stabilization reagent are compared

    Journal: Annals of the New York Academy of Sciences

    Article Title: Genomic Targets in Saliva

    doi: 10.1196/annals.1384.002

    Figure Lengend Snippet: Whole saliva was left untreated or stabilized with RNAlater, Superase or RPS. RNA was extracted immediately or after incubation at room temperature for one week. The transcriptomes of fresh and stored aliquots for each stabilization reagent are compared

    Article Snippet: The preservation of RNA profiles was compared by array analysis from freshly processed sample against the RNA using samples stored at room temperature for one week either with no stabilizer, with Superase Inhibitor (20U/mL, Invitrogen [Carlsbad, CA, USA]), RNAlater (Ambion Inc., Austin, TX, USA; 1:1 mix with saliva) or the RNAprotect Saliva reagent (RPS, Qiagen Inc., Valencia, CA, USA; 1 volume sample plus 5 volumes RPS).

    Techniques: Incubation

    SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor Superase-In (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.

    Journal: Biophysical Journal

    Article Title: SF1 Phosphorylation Enhances Specific Binding to U2AF65 and Reduces Binding to 3′-Splice-Site RNA

    doi: 10.1016/j.bpj.2016.11.007

    Figure Lengend Snippet: SF1 structure and function in pre-mRNA splicing. ( A ) Schematic diagram of the early-stage SF1-U2AF 65 -U2AF 35 splicing-factor complex recognizing the 3′ splice site. ULMs are represented by “W.” White circles represent UHMK1-phosphorylation sites. BPS, branch point sequence; Py, polypyrimidine. ( B ) Domains of SF1, U2AF 65 , and U2AF 35 splicing factors. The boundaries of constructs used in this study are indicated by double-headed arrows. Zn, CCCH zinc knuckle; UHM, U2AF homology motif; ULM, U2AF ligand motif; RRM, RNA recognition motif, RS, arginine-serine repeat; KH-QUA2, K homology with quaking-2 motif; PA, protein A; circled P, UHMK1-phosphorylation sites. ( C ) Structure of the phosphorylated SF1 14–132 -U2AF 65 ). A line representing residues that are absent from the electron density connects the C-terminus of the ULM to the coiled-coil motifs. The tryptophan of the ULM (W22) and phosphorylated serines are shown as spheres and arginine residues that are mutated in this study are shown in stick representation. The inset shows a sequence alignment of the UHMK1-phosphorylated SPSP motif ( bold (between lines)). ( D and E ) Samples of ( D ) phospho-SF1 1–255 and ( E ) AdML L RNA under experimental conditions. Samples either before ( Pre- ) or after ( Post- ) incubating at 23°C for 2 h in the presence of a 10-fold excess of SF1 (U) or phospho-SF1 (P) were analyzed by denaturing gel electrophoresis and stained using ( D ) Coomassie blue or ( E ) SYBR Gold, respectively. The phosphorylated form of SF1 1–255 runs higher by SDS-PAGE. STD, size markers; nt; nucleotides. Conditions were identical to those of fluorescence assays with the exception that the RNase inhibitor Superase-In (Ambion, Life Technologies) was omitted here to fully rule out RNase contamination.

    Article Snippet: Both macromolecules were diluted > 50-fold in 150 mM NaCl, 25 mM HEPES (pH 7.4), 0.2 mM TCEP, and 0.1 U μ L−1 Superase-In (Ambion, Life Technologies, Carlsbad, CA).

    Techniques: Sequencing, Construct, Nucleic Acid Electrophoresis, Staining, SDS Page, Fluorescence

    Trans-protection of mRNA against skin surface ribonuclease activity . Ribonuclease activity assay (negative images). Capped lacZ mRNA in the absence (w/o) or presence of the ribonuclease inhibitors RNasin ® (R-in, final concentration 1 U/μl) or SUPERase· In™ (S-in, 1 U/μl) was incubated with ribonucleases of Homo sapiens hand surface (final dilution 4×) or of Mus musculus ear surface (20×) or with 2, 5 pg/μl ribonuclease A from Bos taurus pancreas at 37°C for increasing time (indicated by the wedge): Samples were taken immediately after addition of ribonucleases as well as 15 min and 60 min after. Samples without ribonucleases are indicated by a dash and were not incubated at 37°C.

    Journal: Genetic Vaccines and Therapy

    Article Title: Characterization of the ribonuclease activity on the skin surface

    doi: 10.1186/1479-0556-4-4

    Figure Lengend Snippet: Trans-protection of mRNA against skin surface ribonuclease activity . Ribonuclease activity assay (negative images). Capped lacZ mRNA in the absence (w/o) or presence of the ribonuclease inhibitors RNasin ® (R-in, final concentration 1 U/μl) or SUPERase· In™ (S-in, 1 U/μl) was incubated with ribonucleases of Homo sapiens hand surface (final dilution 4×) or of Mus musculus ear surface (20×) or with 2, 5 pg/μl ribonuclease A from Bos taurus pancreas at 37°C for increasing time (indicated by the wedge): Samples were taken immediately after addition of ribonucleases as well as 15 min and 60 min after. Samples without ribonucleases are indicated by a dash and were not incubated at 37°C.

    Article Snippet: Recently, a new protein capable of ribonuclease-inhibition was characterized: SUPERase· In™ (Ambion).

    Techniques: Activity Assay, Concentration Assay, Incubation

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Trans-protection of mRNA against skin surface ribonuclease activity . Ribonuclease activity assay (negative images). Capped lacZ mRNA in the absence (w/o) or presence of the ribonuclease inhibitors RNasin ® (R-in, final concentration 1 U/μl) or SUPERase· In™ (S-in, 1 U/μl) was incubated with ribonucleases of Homo sapiens hand surface (final dilution 4×) or of Mus musculus ear surface (20×) or with 2, 5 pg/μl ribonuclease A from Bos taurus pancreas at 37°C for increasing time (indicated by the wedge): Samples were taken immediately after addition of ribonucleases as well as 15 min and 60 min after. Samples without ribonucleases are indicated by a dash and were not incubated at 37°C.

    Journal: Genetic Vaccines and Therapy

    Article Title: Characterization of the ribonuclease activity on the skin surface

    doi: 10.1186/1479-0556-4-4

    Figure Lengend Snippet: Trans-protection of mRNA against skin surface ribonuclease activity . Ribonuclease activity assay (negative images). Capped lacZ mRNA in the absence (w/o) or presence of the ribonuclease inhibitors RNasin ® (R-in, final concentration 1 U/μl) or SUPERase· In™ (S-in, 1 U/μl) was incubated with ribonucleases of Homo sapiens hand surface (final dilution 4×) or of Mus musculus ear surface (20×) or with 2, 5 pg/μl ribonuclease A from Bos taurus pancreas at 37°C for increasing time (indicated by the wedge): Samples were taken immediately after addition of ribonucleases as well as 15 min and 60 min after. Samples without ribonucleases are indicated by a dash and were not incubated at 37°C.

    Article Snippet: Ribonuclease inhibitors The ribonuclease inhibitors RNasin® and SUPERase· In™ were purchased from Promega (Mannheim, Germany) and Ambion (Huntingdon, UK).

    Techniques: Activity Assay, Concentration Assay, Incubation