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  • 93
    Jena Bioscience ara ctp
    Knockout of <t>DCK</t> using a CRISPR‐Cas9 system in KOPN41 cells. (A) Schematic representation of the target sequence of CRISPR‐Cas9. (B) Sequence of the cleavage site in clones #1 and #2. Boxes indicate identical sequences. (C) DCK protein expression in knockout clones and wild‐type (WT) cells as determined by immunoblotting. (D) Intracellular concentrations of <t>Ara‐CTP</t> in wild‐type and DCK knockout (KO) clone of KOPN41. Cells were cultured in the absence or presence of 0.8 (195), 4 (973), and 20 μ mol/L (4864 ng/mL) of Ara‐C for 6 h, and intracellular Ara‐CTP was measured by liquid chromatography–mass spectrometry (LC/MS). The vertical axis indicates intracellular concentration of Ara‐CTP (pmol/1.5 × 10 7 cells), and the horizontal axis indicates the concentration of Ara‐C ( μ mol/L). Error bars indicate standard error of triplicate experiment. Asterisks indicate significance (* P
    Ara Ctp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher biotin 14 ctp
    Purification scheme for CHLAMY 1. (A) Procedure applied for the purification of CHLAMY 1. A <t>CTP-biotinylated</t> transcript ( gs2 wt ) containing the gs2 ), was bound to paramagnetic streptavidin beads according to the procedure described in Materials and Methods. Dialyzed proteins from a 12 to 24% ammonium sulfate precipitation step were incubated with the RNA-bound beads in the presence of poly(G) as nonspecific competitor RNA. After several washing steps, which removed nonbound proteins, CHLAMY 1 was eluted with high salt. A parallel approach with the gs2 mut transcript, which lacks five of the seven UG repeats and cannot be recognized by CHLAMY 1, served as negative control. (B and C) Autoradiograms of mobility shift assays using either the 32 P-labeled gs2 wt transcript or its mutagenized version, gs2 mut. The radiolabeled transcripts do not contain biotinylated nucleotides. For the binding reaction, the samples were incubated in the presence of poly(G) with crude extracts (CE lanes) from Chlamydomonas cells that were harvested at the beginning of the night (LD 12). In addition, dialyzed proteins from a 12 to 24% ammonium sulfate precipitation (AS lanes) of the crude extract mentioned above were also used. RNA-protein complexes were resolved in nondenaturing 4% polyacrylamide gels containing 10% glycerol. (B) With both crude extracts and ammonium sulfate, the wild-type transcript ( gs2 wt ) (lanes 3 and 5) and its mutagenized form ( gs2 mut ) (lanes 4 and 6) were used. Lanes 1 and 2 labeled with RNA demonstrate the mobility of the transcripts ( gs2 wt and gs2 mut ) alone. (C) Autoradiogram of mobility shift assays using the 32 P-labeled gs2 wt transcript in the presence of proteins from the ammonium sulfate step (lane 2). In lanes 3 and 4, a 200× molar excess of unlabeled gs2 wt (lane 3) or gs2 mut (lane 4) transcripts, both containing biotinylated CTP, were added to the binding reaction. Lane 1 shows the mobility of the transcript alone.
    Biotin 14 Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare α 32 p ctp
    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 <t>P]CTP</t> during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.
    α 32 P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies cyanine 3 ctp
    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 <t>P]CTP</t> during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.
    Cyanine 3 Ctp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies cy3 ctp
    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 <t>P]CTP</t> during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.
    Cy3 Ctp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Ctp, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer cyanine 3 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Cyanine 3 Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Ctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Ctp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Agilent technologies cy5 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Cy5 Ctp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer cyanine 5 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Cyanine 5 Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer biotin 11 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Biotin 11 Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare cy5 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Cy5 Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare cy3 ctp
    Effect of Y1699C mutation on LRRK2 <t>GTP</t> binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, <t>CTP,</t> GDP, GTP). B, quantification of the GTP binding assay
    Cy3 Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant α 32 p ctp
    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 <t>P-CTP</t> and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
    α 32 P Ctp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Enzo Biochem bio 11 ctp
    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 <t>P-CTP</t> and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
    Bio 11 Ctp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 88/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies cyanine 3 ctp labeled crna
    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 <t>P-CTP</t> and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
    Cyanine 3 Ctp Labeled Crna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Enzo Biochem biotin ctp
    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 <t>P-CTP</t> and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
    Biotin Ctp, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 89/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Knockout of DCK using a CRISPR‐Cas9 system in KOPN41 cells. (A) Schematic representation of the target sequence of CRISPR‐Cas9. (B) Sequence of the cleavage site in clones #1 and #2. Boxes indicate identical sequences. (C) DCK protein expression in knockout clones and wild‐type (WT) cells as determined by immunoblotting. (D) Intracellular concentrations of Ara‐CTP in wild‐type and DCK knockout (KO) clone of KOPN41. Cells were cultured in the absence or presence of 0.8 (195), 4 (973), and 20 μ mol/L (4864 ng/mL) of Ara‐C for 6 h, and intracellular Ara‐CTP was measured by liquid chromatography–mass spectrometry (LC/MS). The vertical axis indicates intracellular concentration of Ara‐CTP (pmol/1.5 × 10 7 cells), and the horizontal axis indicates the concentration of Ara‐C ( μ mol/L). Error bars indicate standard error of triplicate experiment. Asterisks indicate significance (* P

    Journal: Cancer Medicine

    Article Title: Clofarabine exerts antileukemic activity against cytarabine‐resistant B‐cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression

    doi: 10.1002/cam4.1323

    Figure Lengend Snippet: Knockout of DCK using a CRISPR‐Cas9 system in KOPN41 cells. (A) Schematic representation of the target sequence of CRISPR‐Cas9. (B) Sequence of the cleavage site in clones #1 and #2. Boxes indicate identical sequences. (C) DCK protein expression in knockout clones and wild‐type (WT) cells as determined by immunoblotting. (D) Intracellular concentrations of Ara‐CTP in wild‐type and DCK knockout (KO) clone of KOPN41. Cells were cultured in the absence or presence of 0.8 (195), 4 (973), and 20 μ mol/L (4864 ng/mL) of Ara‐C for 6 h, and intracellular Ara‐CTP was measured by liquid chromatography–mass spectrometry (LC/MS). The vertical axis indicates intracellular concentration of Ara‐CTP (pmol/1.5 × 10 7 cells), and the horizontal axis indicates the concentration of Ara‐C ( μ mol/L). Error bars indicate standard error of triplicate experiment. Asterisks indicate significance (* P

    Article Snippet: We next measured intracellular concentration of Ara‐CTP in both wild‐type and DCK knockout clone (#1) of KOPN41, which were cultured in the absence or presence of 0.8–20 μ mol/L (195–4864 ng/mL) of Ara‐C for 6 h, using liquid chromatography–mass spectrometry (LC/MS).

    Techniques: Knock-Out, CRISPR, Sequencing, Clone Assay, Expressing, Acetylene Reduction Assay, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

    Purification scheme for CHLAMY 1. (A) Procedure applied for the purification of CHLAMY 1. A CTP-biotinylated transcript ( gs2 wt ) containing the gs2 ), was bound to paramagnetic streptavidin beads according to the procedure described in Materials and Methods. Dialyzed proteins from a 12 to 24% ammonium sulfate precipitation step were incubated with the RNA-bound beads in the presence of poly(G) as nonspecific competitor RNA. After several washing steps, which removed nonbound proteins, CHLAMY 1 was eluted with high salt. A parallel approach with the gs2 mut transcript, which lacks five of the seven UG repeats and cannot be recognized by CHLAMY 1, served as negative control. (B and C) Autoradiograms of mobility shift assays using either the 32 P-labeled gs2 wt transcript or its mutagenized version, gs2 mut. The radiolabeled transcripts do not contain biotinylated nucleotides. For the binding reaction, the samples were incubated in the presence of poly(G) with crude extracts (CE lanes) from Chlamydomonas cells that were harvested at the beginning of the night (LD 12). In addition, dialyzed proteins from a 12 to 24% ammonium sulfate precipitation (AS lanes) of the crude extract mentioned above were also used. RNA-protein complexes were resolved in nondenaturing 4% polyacrylamide gels containing 10% glycerol. (B) With both crude extracts and ammonium sulfate, the wild-type transcript ( gs2 wt ) (lanes 3 and 5) and its mutagenized form ( gs2 mut ) (lanes 4 and 6) were used. Lanes 1 and 2 labeled with RNA demonstrate the mobility of the transcripts ( gs2 wt and gs2 mut ) alone. (C) Autoradiogram of mobility shift assays using the 32 P-labeled gs2 wt transcript in the presence of proteins from the ammonium sulfate step (lane 2). In lanes 3 and 4, a 200× molar excess of unlabeled gs2 wt (lane 3) or gs2 mut (lane 4) transcripts, both containing biotinylated CTP, were added to the binding reaction. Lane 1 shows the mobility of the transcript alone.

    Journal: Eukaryotic Cell

    Article Title: The Circadian RNA-Binding Protein CHLAMY 1 Represents a Novel Type Heteromer of RNA Recognition Motif and Lysine Homology Domain-Containing Subunits

    doi: 10.1128/EC.3.3.815-825.2004

    Figure Lengend Snippet: Purification scheme for CHLAMY 1. (A) Procedure applied for the purification of CHLAMY 1. A CTP-biotinylated transcript ( gs2 wt ) containing the gs2 ), was bound to paramagnetic streptavidin beads according to the procedure described in Materials and Methods. Dialyzed proteins from a 12 to 24% ammonium sulfate precipitation step were incubated with the RNA-bound beads in the presence of poly(G) as nonspecific competitor RNA. After several washing steps, which removed nonbound proteins, CHLAMY 1 was eluted with high salt. A parallel approach with the gs2 mut transcript, which lacks five of the seven UG repeats and cannot be recognized by CHLAMY 1, served as negative control. (B and C) Autoradiograms of mobility shift assays using either the 32 P-labeled gs2 wt transcript or its mutagenized version, gs2 mut. The radiolabeled transcripts do not contain biotinylated nucleotides. For the binding reaction, the samples were incubated in the presence of poly(G) with crude extracts (CE lanes) from Chlamydomonas cells that were harvested at the beginning of the night (LD 12). In addition, dialyzed proteins from a 12 to 24% ammonium sulfate precipitation (AS lanes) of the crude extract mentioned above were also used. RNA-protein complexes were resolved in nondenaturing 4% polyacrylamide gels containing 10% glycerol. (B) With both crude extracts and ammonium sulfate, the wild-type transcript ( gs2 wt ) (lanes 3 and 5) and its mutagenized form ( gs2 mut ) (lanes 4 and 6) were used. Lanes 1 and 2 labeled with RNA demonstrate the mobility of the transcripts ( gs2 wt and gs2 mut ) alone. (C) Autoradiogram of mobility shift assays using the 32 P-labeled gs2 wt transcript in the presence of proteins from the ammonium sulfate step (lane 2). In lanes 3 and 4, a 200× molar excess of unlabeled gs2 wt (lane 3) or gs2 mut (lane 4) transcripts, both containing biotinylated CTP, were added to the binding reaction. Lane 1 shows the mobility of the transcript alone.

    Article Snippet: Then, 20 μg of biotinylated transcript was synthesized in the presence of biotinylated CTP (biotin-14-CTP; Gibco BRL) in addition to the unmodified nucleotides by a procedure described before ( ).

    Techniques: Purification, Incubation, Negative Control, Mobility Shift, Labeling, Binding Assay

    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 P]CTP during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 P]CTP during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro, Binding Assay, Concentration Assay, Activity Assay, Labeling

    In vitro transcription assays with the hly and actA promoters of L. monocytogenes (P hly Lm and P actA Lm ), L. ivanovii (i-P hly and i-P actA ), and L. seeligeri (s-P hly and s-P actA ). Results are from an in vitro transcription assay with increasing concentrations of the PrfA proteins PrfA Lm , PrfA* Lm , PrfA Ls , and PrfA Li using 16 nM promoter template DNA and 1.3 nM (when using P hly ) or 1.9 nM (when using P actA ) RNA polymerase. The runoff transcripts were radioactively marked by adding [α- 32 P]CTP to the in vitro assay. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. Values represent the relative ratios of the measured radioactivities and the molar concentrations of PrfA protein in the range of linear dependency. The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: In vitro transcription assays with the hly and actA promoters of L. monocytogenes (P hly Lm and P actA Lm ), L. ivanovii (i-P hly and i-P actA ), and L. seeligeri (s-P hly and s-P actA ). Results are from an in vitro transcription assay with increasing concentrations of the PrfA proteins PrfA Lm , PrfA* Lm , PrfA Ls , and PrfA Li using 16 nM promoter template DNA and 1.3 nM (when using P hly ) or 1.9 nM (when using P actA ) RNA polymerase. The runoff transcripts were radioactively marked by adding [α- 32 P]CTP to the in vitro assay. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. Values represent the relative ratios of the measured radioactivities and the molar concentrations of PrfA protein in the range of linear dependency. The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro

    Replacement of the C-terminal 38 amino acids of PrfA Ls with those of PrfA Lm . (A) ClustalW alignment of the PrfA proteins of L. monocytogenes (PrfA Lm ), L. ivanovii (PrfA Li ), and L. seeligeri (PrfA Ls ). Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Amino acid substitutions leading to a constitutively active PrfA are marked. (B) In vitro transcription assay with increasing concentrations of PrfA Lm , the hybrid PrfA Lsm , and PrfA Ls using 16 nM promoter template DNA and 1.9 nM RNA polymerase. The mRNA was marked with [α- 32 P]CTP during transcription. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. The graphs in the lower part of the panel below show the amounts of CI and CII measured in EMSAs. The components were 5 nM 32 P-marked promoter DNA (P hly Lm ) and 1.5 nM RNA polymerase, and the PrfA concentration is given in the figure. Quantification of CI and CII complexes was performed using ImageMaster (Amersham). The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: Replacement of the C-terminal 38 amino acids of PrfA Ls with those of PrfA Lm . (A) ClustalW alignment of the PrfA proteins of L. monocytogenes (PrfA Lm ), L. ivanovii (PrfA Li ), and L. seeligeri (PrfA Ls ). Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Amino acid substitutions leading to a constitutively active PrfA are marked. (B) In vitro transcription assay with increasing concentrations of PrfA Lm , the hybrid PrfA Lsm , and PrfA Ls using 16 nM promoter template DNA and 1.9 nM RNA polymerase. The mRNA was marked with [α- 32 P]CTP during transcription. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. The graphs in the lower part of the panel below show the amounts of CI and CII measured in EMSAs. The components were 5 nM 32 P-marked promoter DNA (P hly Lm ) and 1.5 nM RNA polymerase, and the PrfA concentration is given in the figure. Quantification of CI and CII complexes was performed using ImageMaster (Amersham). The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro, Concentration Assay

    In situ localization of ILK mRNA in the skin of the transgenic mouse overexpressing the activated erbB-2. A to D: In situ localization of ILK and erbB-2 mRNAs. Frozen sections of the back skin of a 1-day-old K14-erbB-2 transgenic founder mouse (TG4839) were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled cRNA probes for the ILK ( A and B ) or the erbB-2 ( C and D ). After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. E and F: PCNA immunostaining in neonatal skins of normal ( E ) and the K14-erbB-2 ( F ) mice. Paraffin sections were subjected to PCNA immunostaining with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen. The sections were counterstained with hematoxylin. Original magnification ( A to F ), ×200.

    Journal: The American Journal of Pathology

    Article Title: Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

    doi:

    Figure Lengend Snippet: In situ localization of ILK mRNA in the skin of the transgenic mouse overexpressing the activated erbB-2. A to D: In situ localization of ILK and erbB-2 mRNAs. Frozen sections of the back skin of a 1-day-old K14-erbB-2 transgenic founder mouse (TG4839) were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled cRNA probes for the ILK ( A and B ) or the erbB-2 ( C and D ). After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. E and F: PCNA immunostaining in neonatal skins of normal ( E ) and the K14-erbB-2 ( F ) mice. Paraffin sections were subjected to PCNA immunostaining with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen. The sections were counterstained with hematoxylin. Original magnification ( A to F ), ×200.

    Article Snippet: High-specific activity riboprobes were synthesized in 10-μl reactions containing 100 μCi [35 S]UTP and 100 μCi [35 S]CTP (Amersham, Arlington Heights, IL), 10 mmol/L NaCl, 6 mmol/L MgCl2 , 40 mmol/L Tris (ph 7.5), 2 mmol/L spermidine, 10 mmol/L dithiothreitol, 500 μmol/L each of unlabeled ATP and GTP, 25 μmol/L each of unlabeled UTP and CTP, 0.5–1 μg linearized template, 15 U of the appropriate polymerase, and 15 U RNase inhibitor (RNasin, Promega, Madison, WI).

    Techniques: In Situ, Transgenic Assay, In Situ Hybridization, Labeling, Hybridization, Microscopy, Immunostaining, Mouse Assay

    In situ localization of ILK and PINCH mRNAs in normal mouse skins. Frozen sections of the back skin of 1-day-old mouse were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled ILK ( A and B ) and PINCH ( C and D ) cRNA probes, respectively. After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. SC, stratum corneum; GR, granular cells; SP, spinous cells; BL, basal layer; hf, hair follicle. Arrows indicate hair follicles. Original magnification ( A to D ), ×200.

    Journal: The American Journal of Pathology

    Article Title: Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

    doi:

    Figure Lengend Snippet: In situ localization of ILK and PINCH mRNAs in normal mouse skins. Frozen sections of the back skin of 1-day-old mouse were subjected to in situ hybridization using [ 35 S]UTP- and [ 35 S]CTP-labeled ILK ( A and B ) and PINCH ( C and D ) cRNA probes, respectively. After hybridization, sections were dipped in liquid emulsion and exposed for 1 week before developing. The sections were counterstained with hematoxylin and eosin. The same fields were examined by bright-field ( A and C ) and dark-field ( B and D ) microscopy. SC, stratum corneum; GR, granular cells; SP, spinous cells; BL, basal layer; hf, hair follicle. Arrows indicate hair follicles. Original magnification ( A to D ), ×200.

    Article Snippet: High-specific activity riboprobes were synthesized in 10-μl reactions containing 100 μCi [35 S]UTP and 100 μCi [35 S]CTP (Amersham, Arlington Heights, IL), 10 mmol/L NaCl, 6 mmol/L MgCl2 , 40 mmol/L Tris (ph 7.5), 2 mmol/L spermidine, 10 mmol/L dithiothreitol, 500 μmol/L each of unlabeled ATP and GTP, 25 μmol/L each of unlabeled UTP and CTP, 0.5–1 μg linearized template, 15 U of the appropriate polymerase, and 15 U RNase inhibitor (RNasin, Promega, Madison, WI).

    Techniques: In Situ, In Situ Hybridization, Labeling, Hybridization, Microscopy

    CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Journal:

    Article Title: Molecular basis for maintenance of fidelity during the CCA-adding reaction by a CCA-adding enzyme

    doi: 10.1038/emboj.2008.124

    Figure Lengend Snippet: CTP and ATP incorporation into mutant mini-helix variants. ( A ) CMP incorporation into mini-D 73 N 74 in the presence of α- 32 P CTP and unlabelled ATP by the wild-type AFCCA (left part) or the R 224 A mutant AFCCA (right part). The upper panel shows

    Article Snippet: The assay procedures for CMP incorporation into mini-D73 N74 were the same as those described above, except that 100 μM CTP, 100 μM ATP, and 100 nM α-32 P CTP (3000 Ci/mmol; GE Healthcare) were used instead of the 100 μM ATP and 100 nM α-32 P ATP.

    Techniques: Mutagenesis

    Effect of Y1699C mutation on LRRK2 GTP binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, CTP, GDP, GTP). B, quantification of the GTP binding assay

    Journal: Journal of neurochemistry

    Article Title: INSIGHT INTO THE MODE OF ACTION OF THE LRRK2 Y1699C PATHOGENIC MUTANT

    doi: 10.1111/j.1471-4159.2010.07105.x

    Figure Lengend Snippet: Effect of Y1699C mutation on LRRK2 GTP binding capacity. A, Flag M2 immuno blot indicating input and GTP-bound LRRK2 levels in the absence (−) or presence of competing nucleotides (ATP, CTP, GDP, GTP). B, quantification of the GTP binding assay

    Article Snippet: Afterwards, nucleotides, i.e. GTP, GDP, ATP or CTP (all Sigma), were added to a final concentration of 2 mM, and the incubation was continued for another 60 min at 4°C.

    Techniques: Mutagenesis, Binding Assay, GTP Binding Assay

    The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 P-CTP and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.

    Journal: PLoS ONE

    Article Title: Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

    doi: 10.1371/journal.pone.0025837

    Figure Lengend Snippet: The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 P-CTP and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.

    Article Snippet: RdRp assays RdRp assays were performed as 20 µL reactions containing 20 mM sodium glutamate (pH 8.2), 12.5 mM DTT, 4 mM MgCl2 , 1 mM MnCl2 , 0.5% Triton X-100, 0.2 mM GTP, 0.1 mM ATP and UTP, 250 nM [α-32 P] CTP (MP Biomedicals), 2 pmol PE46 and 1 pmol LE19p as templates .

    Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Labeling, Electrophoretic Mobility Shift Assay