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Figure 1. DC-SIGN Signaling Modulates TLR3-, TLR4-, and TLR5-Dependent IL-10 Induction (A) ManLAM induces IL-10 after TLR3, TLR4, and TLR5 triggering. Human iDCs were treated with TLR4 ligand LPS, TLR3 ligand poly(I:C), TLR5 ligand flagellin, ManLAM, or combinations, and IL-10 protein amounts were measured by ELISA after 24 hr. A representative experiment of two donors is shown. Results are presented as the means ± SD from duplicate samples. (B) IL-10 <t>mRNA</t> production is also increased after TLR activation. Cells were treated similar as in (A). Specificity for DC-SIGN was determined by preincubating the cells for 2 hr with the blocking anti-DC-SIGN antibody AZN-D2. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative <t>real-time</t> <t>PCR</t> analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (C) ManLAM does not affect IL-10 mRNA stability after LPS stimulation. Cells were stimulated as indicated for 3 hr, before the addition of 10 mg/ml actinomycin D (ActD) to block transcription. At 20 min time periods, mRNA expression was quantified by real-time PCR analysis.
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Figure 1. DC-SIGN Signaling Modulates TLR3-, TLR4-, and TLR5-Dependent IL-10 Induction (A) ManLAM induces IL-10 after TLR3, TLR4, and TLR5 triggering. Human iDCs were treated with TLR4 ligand LPS, TLR3 ligand poly(I:C), TLR5 ligand flagellin, ManLAM, or combinations, and IL-10 protein amounts were measured by ELISA after 24 hr. A representative experiment of two donors is shown. Results are presented as the means ± SD from duplicate samples. (B) IL-10 mRNA production is also increased after TLR activation. Cells were treated similar as in (A). Specificity for DC-SIGN was determined by preincubating the cells for 2 hr with the blocking anti-DC-SIGN antibody AZN-D2. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (C) ManLAM does not affect IL-10 mRNA stability after LPS stimulation. Cells were stimulated as indicated for 3 hr, before the addition of 10 mg/ml actinomycin D (ActD) to block transcription. At 20 min time periods, mRNA expression was quantified by real-time PCR analysis.

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 1. DC-SIGN Signaling Modulates TLR3-, TLR4-, and TLR5-Dependent IL-10 Induction (A) ManLAM induces IL-10 after TLR3, TLR4, and TLR5 triggering. Human iDCs were treated with TLR4 ligand LPS, TLR3 ligand poly(I:C), TLR5 ligand flagellin, ManLAM, or combinations, and IL-10 protein amounts were measured by ELISA after 24 hr. A representative experiment of two donors is shown. Results are presented as the means ± SD from duplicate samples. (B) IL-10 mRNA production is also increased after TLR activation. Cells were treated similar as in (A). Specificity for DC-SIGN was determined by preincubating the cells for 2 hr with the blocking anti-DC-SIGN antibody AZN-D2. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (C) ManLAM does not affect IL-10 mRNA stability after LPS stimulation. Cells were stimulated as indicated for 3 hr, before the addition of 10 mg/ml actinomycin D (ActD) to block transcription. At 20 min time periods, mRNA expression was quantified by real-time PCR analysis.

Article Snippet: Quantitative Real-Time PCR and mRNA Half-Life Determination mRNA was specifically isolated with the mRNA capture kit (Roche, Basel, Switzerland), and cDNA was synthesized with the Reverse transcriptase kit (Promega, Madison, WI).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Blocking Assay, Expressing, Real-time Polymerase Chain Reaction

Figure 2. DC-SIGN-Mediated Modula- tion of TLR-Dependent IL-10 Induction Requires Raf Kinase Activity (A) Relative IL-10 mRNA expression by human iDCs treated with LPS and/or ManLAM for 6 hr in the presence of either Raf inhibitor GW5074, NF-kB inhibitor BAY11-7082, Syk inhibitor pi- ceatannol, p38 inhibitor SB203580, MEK1/2 in- hibitor U0126, PI 3-kinase inhibitor LY294002, PKB inhibitor API-2, PKA/MSK1 inhibitor H89, or 0.1% DMSO as a control; cells were prein- cubated for 2 hr with inhibitors before stimuli were added. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (B) Inhibition of Raf-1 blocks IL-10 upregulation induced by ManLAM after stimulation of DCs with different TLR ligands. Cells were stimu- lated with ligands and Raf inhibitor GW5074 as in (A). Results are presented as the means ± SD from at least three independent experiments.

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 2. DC-SIGN-Mediated Modula- tion of TLR-Dependent IL-10 Induction Requires Raf Kinase Activity (A) Relative IL-10 mRNA expression by human iDCs treated with LPS and/or ManLAM for 6 hr in the presence of either Raf inhibitor GW5074, NF-kB inhibitor BAY11-7082, Syk inhibitor pi- ceatannol, p38 inhibitor SB203580, MEK1/2 in- hibitor U0126, PI 3-kinase inhibitor LY294002, PKB inhibitor API-2, PKA/MSK1 inhibitor H89, or 0.1% DMSO as a control; cells were prein- cubated for 2 hr with inhibitors before stimuli were added. After 6 hr of stimulation, relative IL-10 mRNA expression was determined by quantitative real-time PCR analysis. IL-10 mRNA expression in LPS-stimulated cells was set at 1. Results are presented as the means ± SD from at least three independent experiments. (B) Inhibition of Raf-1 blocks IL-10 upregulation induced by ManLAM after stimulation of DCs with different TLR ligands. Cells were stimu- lated with ligands and Raf inhibitor GW5074 as in (A). Results are presented as the means ± SD from at least three independent experiments.

Article Snippet: Quantitative Real-Time PCR and mRNA Half-Life Determination mRNA was specifically isolated with the mRNA capture kit (Roche, Basel, Switzerland), and cDNA was synthesized with the Reverse transcriptase kit (Promega, Madison, WI).

Techniques: Activity Assay, Expressing, Control, Real-time Polymerase Chain Reaction, Inhibition

Figure 3. DC-SIGN Signaling Induces Raf-1 Activation, Independent of TLR4 Signaling (A–C) Kinase activity assay of B-Raf (A)-, Raf-1 (B)-, or ERK1/2 (C)-specific immunocomplexes from cell lysates of human iDCs treated with LPS or DC-SIGN ligand ManLAM for 10 min; cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indicated. Kinase activities in either LPS (A, C)- or ManLAM (B)-stimulated cells were set at 1. Results are presented as the means ± SD from three independent experiments. (D) Raf-1 was specifically silenced by RNA interference. DCs were transfected with 100 nM Raf-1 SMARTpool or control nontargeting siRNAs. At 72 hr after transfection, B-Raf and Raf-1 mRNA was measured by quantitative real-time PCR. Results are presented as the means ± SD from two indepen- dent experiments. (E)Raf-1isessentialtoManLAM-inducedIL-10upregulation.Raf-1orcontrolsiRNAknockdownDCswereincubatedwithLPSaloneorincombinationwith ManLAM, and IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from two independent experiments.

Journal: Immunity

Article Title: C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

doi: 10.1016/j.immuni.2007.03.012

Figure Lengend Snippet: Figure 3. DC-SIGN Signaling Induces Raf-1 Activation, Independent of TLR4 Signaling (A–C) Kinase activity assay of B-Raf (A)-, Raf-1 (B)-, or ERK1/2 (C)-specific immunocomplexes from cell lysates of human iDCs treated with LPS or DC-SIGN ligand ManLAM for 10 min; cells were preincubated for 2 hr with the blocking DC-SIGN antibody AZN-D2 as indicated. Kinase activities in either LPS (A, C)- or ManLAM (B)-stimulated cells were set at 1. Results are presented as the means ± SD from three independent experiments. (D) Raf-1 was specifically silenced by RNA interference. DCs were transfected with 100 nM Raf-1 SMARTpool or control nontargeting siRNAs. At 72 hr after transfection, B-Raf and Raf-1 mRNA was measured by quantitative real-time PCR. Results are presented as the means ± SD from two indepen- dent experiments. (E)Raf-1isessentialtoManLAM-inducedIL-10upregulation.Raf-1orcontrolsiRNAknockdownDCswereincubatedwithLPSaloneorincombinationwith ManLAM, and IL-10 mRNA was determined as described in Figure 1A. Results are presented as the means ± SD from two independent experiments.

Article Snippet: Quantitative Real-Time PCR and mRNA Half-Life Determination mRNA was specifically isolated with the mRNA capture kit (Roche, Basel, Switzerland), and cDNA was synthesized with the Reverse transcriptase kit (Promega, Madison, WI).

Techniques: Activation Assay, Kinase Assay, Blocking Assay, Transfection, Control, Real-time Polymerase Chain Reaction