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Promega superscriptii reverse transcriptase
Superscriptii Reverse Transcriptase, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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superscriptii reverse transcriptase - by Bioz Stars, 2020-04
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Isolation:

Article Title: The RNA binding protein Musashi1 regulates apoptosis, gene expression and stress granule formation in urothelial carcinoma cells
Article Snippet: .. RNA extraction Total RNA was isolated from sub-confluent cell cultures and cell lines using Qiazol reagent (Qiagen, Hilden, Germany) and purified via RNeasy columns (Qiagen). cDNA synthesis was performed with SuperScriptII reverse transcriptase (Promega, Mannheim, Germany) with oligo-dT primers as described [ ]. .. DNA extraction High molecular weight genomic DNA from cell lines was isolated using the blood and cell culture DNA kit (Qiagen) with additional proteinase K treatment.

RNA Extraction:

Article Title: The RNA binding protein Musashi1 regulates apoptosis, gene expression and stress granule formation in urothelial carcinoma cells
Article Snippet: .. RNA extraction Total RNA was isolated from sub-confluent cell cultures and cell lines using Qiazol reagent (Qiagen, Hilden, Germany) and purified via RNeasy columns (Qiagen). cDNA synthesis was performed with SuperScriptII reverse transcriptase (Promega, Mannheim, Germany) with oligo-dT primers as described [ ]. .. DNA extraction High molecular weight genomic DNA from cell lines was isolated using the blood and cell culture DNA kit (Qiagen) with additional proteinase K treatment.

Purification:

Article Title: The RNA binding protein Musashi1 regulates apoptosis, gene expression and stress granule formation in urothelial carcinoma cells
Article Snippet: .. RNA extraction Total RNA was isolated from sub-confluent cell cultures and cell lines using Qiazol reagent (Qiagen, Hilden, Germany) and purified via RNeasy columns (Qiagen). cDNA synthesis was performed with SuperScriptII reverse transcriptase (Promega, Mannheim, Germany) with oligo-dT primers as described [ ]. .. DNA extraction High molecular weight genomic DNA from cell lines was isolated using the blood and cell culture DNA kit (Qiagen) with additional proteinase K treatment.

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  • 85
    Promega bgl i
    Check of the integrity of codon 634 with the <t>Bgl</t> I enzyme. DNA samples were amplified using forward and reverse primers shown in figure 1 . The PCR product was incubated in the presence (lane 2–5) or absence (lane 1) of Bgl I. The restriction fragments were run in a non-denaturing 10% PAGE. Lane 1: wild type 634 codon without enzyme incubation ; lane 2: wild type 634 codon ; lane 3: mutation in the first base of the 634 codon (T > C); lane 4: mutation in the second base of the 634 codon (G > A); lane 5: mutation in the third base of the 634 codon (C > G). Both alleles are completely cut by the enzyme in lane 2 and 3, which means that at least the second and third bases of the codon are correct. In lane 4 and 5, the product is not completely cut, which reveals that one allele has a mutation at the second or third base of codon 634.
    Bgl I, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bgl i/product/Promega
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    97
    Promega ifn γ
    Diagram of the proposed signaling pathways that modulate ManLAM-induced iNOS expression. The required <t>IFN-γ</t> costimulus pathway utilizing transcription factors IRF-1 and Stat1α is also shown.
    Ifn γ, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega protein kinase a pka
    Measurements of CFTR conductance in inside-out patch clamp recordings with recombinant WNK1 proteins. ( A , B ) Macroscopic currents were recorded from inside-out membrane patches using symmetrically Cl − -rich (150 mM) solutions in CFTR-expressing HEK293T cells. Patch membranes were prepared from HEK293T cells expressing CFTR. CFTR currents were activated by addition of the catalytic subunit of protein <t>kinase</t> A <t>(PKA,</t> 10 unit/mL) to a 3-mM MgATP containing bath solution (intracellular side). The I-V relationship was obtained by applying ramp pulses from −80 to +80 mV (0.8 mV/ms) at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel A ) and a summary of relative CFTR currents at –60 mV (normalized by control G Cl ) are shown in panel B (n = 10). ( C , D ) Macroscopic CFTR currents were recorded from inside-out membrane patches using symmetrically HCO 3 – -rich (146 HCO 3 – + 4 Cl – ) solutions. The I-V relationship was obtained at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel C and a summary of relative CFTR currents (normalized by control G HCO3 ) are shown in panel D (n = 9). Data are presented as the mean ± SEM. ** P
    Protein Kinase A Pka, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega superscript iii reverse transcriptase
    miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in <t>three</t> hepatoma-derived cell lines transfected with JFH1 <t>RNA.</t> ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p
    Superscript Iii Reverse Transcriptase, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Check of the integrity of codon 634 with the Bgl I enzyme. DNA samples were amplified using forward and reverse primers shown in figure 1 . The PCR product was incubated in the presence (lane 2–5) or absence (lane 1) of Bgl I. The restriction fragments were run in a non-denaturing 10% PAGE. Lane 1: wild type 634 codon without enzyme incubation ; lane 2: wild type 634 codon ; lane 3: mutation in the first base of the 634 codon (T > C); lane 4: mutation in the second base of the 634 codon (G > A); lane 5: mutation in the third base of the 634 codon (C > G). Both alleles are completely cut by the enzyme in lane 2 and 3, which means that at least the second and third bases of the codon are correct. In lane 4 and 5, the product is not completely cut, which reveals that one allele has a mutation at the second or third base of codon 634.

    Journal: BMC Medical Genetics

    Article Title: A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A.

    doi: 10.1186/1471-2350-3-4

    Figure Lengend Snippet: Check of the integrity of codon 634 with the Bgl I enzyme. DNA samples were amplified using forward and reverse primers shown in figure 1 . The PCR product was incubated in the presence (lane 2–5) or absence (lane 1) of Bgl I. The restriction fragments were run in a non-denaturing 10% PAGE. Lane 1: wild type 634 codon without enzyme incubation ; lane 2: wild type 634 codon ; lane 3: mutation in the first base of the 634 codon (T > C); lane 4: mutation in the second base of the 634 codon (G > A); lane 5: mutation in the third base of the 634 codon (C > G). Both alleles are completely cut by the enzyme in lane 2 and 3, which means that at least the second and third bases of the codon are correct. In lane 4 and 5, the product is not completely cut, which reveals that one allele has a mutation at the second or third base of codon 634.

    Article Snippet: PCR products were incubated with 0.5 μl of Bst ApI (10 U/μl New England Biolabs, Buenos Aires, Argentina) or Bgl I (10 U/μl Promega, Buenos Aires, Argentina), in a 25 μl reaction volume.

    Techniques: Amplification, Polymerase Chain Reaction, Incubation, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Diagram of the proposed signaling pathways that modulate ManLAM-induced iNOS expression. The required IFN-γ costimulus pathway utilizing transcription factors IRF-1 and Stat1α is also shown.

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: Diagram of the proposed signaling pathways that modulate ManLAM-induced iNOS expression. The required IFN-γ costimulus pathway utilizing transcription factors IRF-1 and Stat1α is also shown.

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Expressing

    The effects of NF-κB inhibition on NO 2 − expression. Macrophages were pretreated with BAY 11-7082 (1 to 10 μM) or DMSO (0.02%) for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by NO 2 − assay. Data shown are the means ± SD of three independent experiments.

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: The effects of NF-κB inhibition on NO 2 − expression. Macrophages were pretreated with BAY 11-7082 (1 to 10 μM) or DMSO (0.02%) for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by NO 2 − assay. Data shown are the means ± SD of three independent experiments.

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Inhibition, Expressing

    IFN-γ synergizes with ManLAM in iNOS and NO 2 − expression. (A) RAW 264.7γNO(−) cells were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or combined IFN-γ (10 U/ml) and ManLAM (0.01 to 10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO 2 − in the supernatant. Data shown are the means ± SD of three independent experiments. (B) Macrophages were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or both for 18 h, and nucleus-free lysates were separated by SDS-PAGE and immunoblotted with a polyclonal iNOS antibody. The immunoblot shown is representative of three independent experiments.

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: IFN-γ synergizes with ManLAM in iNOS and NO 2 − expression. (A) RAW 264.7γNO(−) cells were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or combined IFN-γ (10 U/ml) and ManLAM (0.01 to 10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO 2 − in the supernatant. Data shown are the means ± SD of three independent experiments. (B) Macrophages were stimulated with IFN-γ (10 U/ml), ManLAM (10 μg/ml), or both for 18 h, and nucleus-free lysates were separated by SDS-PAGE and immunoblotted with a polyclonal iNOS antibody. The immunoblot shown is representative of three independent experiments.

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Expressing, SDS Page

    Inhibition of MEK1-ERK by PD98059 inhibits iNOS and NO 2 − expression by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were pretreated with various concentrations of PD98059 (0.1 to 30 μM) or with vehicle DMSO (D) at a concentration of 0.075% for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO 2 − . Data shown are the means ± SD of three independent experiments. ∗∗∗, P

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: Inhibition of MEK1-ERK by PD98059 inhibits iNOS and NO 2 − expression by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were pretreated with various concentrations of PD98059 (0.1 to 30 μM) or with vehicle DMSO (D) at a concentration of 0.075% for 1 h and then costimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 18 h, followed by the Greiss reagent assay for NO 2 − . Data shown are the means ± SD of three independent experiments. ∗∗∗, P

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Inhibition, Expressing, Concentration Assay

    Inhibition of p38 mapk with SB203580 augmented iNOS and NO 2 − expression stimulated by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were cultured with media alone, IFN-γ (10 U/ml) plus SB203580 (30 μM), IFN-γ (10 U/ml) plus ManLAM (10 μg/ml), or IFN-γ plus ManLAM plus SB203580. The cells were preincubated with SB203580 for 1 h and then cocultured with the indicated stimuli. After 18 h of stimulation, the supernatant was assayed for NO 2 − . Data shown are the means ± SD of three independent experiments. ∗∗∗, P

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: Inhibition of p38 mapk with SB203580 augmented iNOS and NO 2 − expression stimulated by IFN-γ plus ManLAM. (A) RAW 264.7γNO(−) macrophages were cultured with media alone, IFN-γ (10 U/ml) plus SB203580 (30 μM), IFN-γ (10 U/ml) plus ManLAM (10 μg/ml), or IFN-γ plus ManLAM plus SB203580. The cells were preincubated with SB203580 for 1 h and then cocultured with the indicated stimuli. After 18 h of stimulation, the supernatant was assayed for NO 2 − . Data shown are the means ± SD of three independent experiments. ∗∗∗, P

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Inhibition, Expressing, Cell Culture

    The effects of a mutant (null) IκBα on IFN-γ–ManLAM-induced iNOS promoter activity. RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 1 μg of null IκBα plasmid or 1 μg of pCMV5 vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). Nucleus-free lysates were then measured for luciferase activity after 8 h of stimulation. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗, P

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: The effects of a mutant (null) IκBα on IFN-γ–ManLAM-induced iNOS promoter activity. RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 1 μg of null IκBα plasmid or 1 μg of pCMV5 vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). Nucleus-free lysates were then measured for luciferase activity after 8 h of stimulation. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗, P

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Mutagenesis, Activity Assay, Plasmid Preparation, Luciferase, Protein Concentration

    The effects of DN-MKK7 and DN-MKK4 on IFN-γ–ManLAM-induced iNOS-luc activity. (A) RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 2 μg of DN-MKK7 plasmid or 2 μg of pCMV5 empty expression vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). After 8 h of stimulation, the cells were lysed and the nucleus-free lysates were then measured for luciferase activity. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗∗, P

    Journal: Infection and Immunity

    Article Title: Induction of Inducible Nitric Oxide Synthase-NO? by Lipoarabinomannan of Mycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-?B Signaling Pathways

    doi: 10.1128/IAI.69.4.2001-2010.2001

    Figure Lengend Snippet: The effects of DN-MKK7 and DN-MKK4 on IFN-γ–ManLAM-induced iNOS-luc activity. (A) RAW 264.7γNO(−) macrophages were cotransfected with 0.3 μg of iNOS-luc plasmid and 2 μg of DN-MKK7 plasmid or 2 μg of pCMV5 empty expression vector, followed by stimulation with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml). After 8 h of stimulation, the cells were lysed and the nucleus-free lysates were then measured for luciferase activity. The results are reported as fold increases in relative light units (fold RLU) and are normalized for protein concentration. Data shown are the means ± SD of three independent experiments. ∗∗, P

    Article Snippet: The media were changed 24 h after transfection, and, after an additional 48 h, the cells were stimulated with IFN-γ (10 U/ml) plus ManLAM (10 μg/ml) for 8 h. The cells were then washed with phosphate-buffered saline, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions (Promega Inc.).

    Techniques: Activity Assay, Plasmid Preparation, Expressing, Luciferase, Protein Concentration

    Measurements of CFTR conductance in inside-out patch clamp recordings with recombinant WNK1 proteins. ( A , B ) Macroscopic currents were recorded from inside-out membrane patches using symmetrically Cl − -rich (150 mM) solutions in CFTR-expressing HEK293T cells. Patch membranes were prepared from HEK293T cells expressing CFTR. CFTR currents were activated by addition of the catalytic subunit of protein kinase A (PKA, 10 unit/mL) to a 3-mM MgATP containing bath solution (intracellular side). The I-V relationship was obtained by applying ramp pulses from −80 to +80 mV (0.8 mV/ms) at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel A ) and a summary of relative CFTR currents at –60 mV (normalized by control G Cl ) are shown in panel B (n = 10). ( C , D ) Macroscopic CFTR currents were recorded from inside-out membrane patches using symmetrically HCO 3 – -rich (146 HCO 3 – + 4 Cl – ) solutions. The I-V relationship was obtained at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel C and a summary of relative CFTR currents (normalized by control G HCO3 ) are shown in panel D (n = 9). Data are presented as the mean ± SEM. ** P

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders

    doi: 10.1016/j.jcmgh.2019.09.003

    Figure Lengend Snippet: Measurements of CFTR conductance in inside-out patch clamp recordings with recombinant WNK1 proteins. ( A , B ) Macroscopic currents were recorded from inside-out membrane patches using symmetrically Cl − -rich (150 mM) solutions in CFTR-expressing HEK293T cells. Patch membranes were prepared from HEK293T cells expressing CFTR. CFTR currents were activated by addition of the catalytic subunit of protein kinase A (PKA, 10 unit/mL) to a 3-mM MgATP containing bath solution (intracellular side). The I-V relationship was obtained by applying ramp pulses from −80 to +80 mV (0.8 mV/ms) at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel A ) and a summary of relative CFTR currents at –60 mV (normalized by control G Cl ) are shown in panel B (n = 10). ( C , D ) Macroscopic CFTR currents were recorded from inside-out membrane patches using symmetrically HCO 3 – -rich (146 HCO 3 – + 4 Cl – ) solutions. The I-V relationship was obtained at resting state (basal) and after additions of PKA (Control) and PKA + WNK1(1–491) to the bath solution. Representative traces are shown in panel C and a summary of relative CFTR currents (normalized by control G HCO3 ) are shown in panel D (n = 9). Data are presented as the mean ± SEM. ** P

    Article Snippet: The ATP (3 mM)-containing pipette solutions also contained the catalytic subunit of protein kinase A (PKA) (10 U/mL; Promega, Madison, WI, USA) to activate CFTR currents.

    Techniques: Patch Clamp, Recombinant, Expressing, Mass Spectrometry

    miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: miR-122 promotes antiviral IFN response. ( A ) Western blot analysis of p-STAT1 and MDA5 in three hepatoma-derived cell lines transfected with JFH1 RNA. ( B ) Comparison of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. ( C ) qRT-PCR analysis of HBV pgRNA levels in HepG2-2.15 (G2-2.15), HepG2 cells transfected with HBV 1.3-mer vector (G2-1.3), HepG2 cells transfected with total RNAs isolated from HepG2-2.15 (HBV2.15) or G2-1.3 (HBV1.3). ( D ) qRT-PCR analysis of IFNs in HepG2 cells first transfected with miR-NC or miR-122 for 2 days, and then treated with the indicated nucleic acids for 24 hr. ( E ) qRT-PCR analysis of the indicated genes in HepG2 cells treated with mimics and then JFH1, as in panel D. ( F ) Analysis of the IFN mRNAs and p-STAT1 in Huh7 cells first transfected with miR-NC or miR-122 mimics and then treated with JFH1. qRT-PCR data are one experiment representative of two ( B and C ) or three ( D–F ) independent experiments (mean ±SEM). *p

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Western Blot, Derivative Assay, Transfection, Expressing, Quantitative RT-PCR, Plasmid Preparation, Isolation

    STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: STAT3 inhibits the transcriptional activation of IRF1. ( A ) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. ( B ) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. ( C ) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). ( D ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. ( F ) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. ( G ) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Activation Assay, Quantitative RT-PCR, Transfection, Expressing, Negative Control

    The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: The effect of miR-122 on HCV translation. ( A ) Structure of pSGR-JFH1/Gluc constructs. ( B ) Luciferase assays of the activity of Gluc reporter treated with miR-122 mimic or XRN1 siRNA. HepG2 cells were firstly treated with mimics or siRNAs for 48 hr and then transfected with SGR-JFH1/Gluc RNA for 24 hr. The activities of Gluc were measured and normalized to the level in miR-NC-treated cells. Data shown are mean +SD (n = 3). Gluc data are one experiment representative of three independent experiments (mean ± SEM of technical triplicates). ( C ) Comparing the effect of miR-122 on the translation of wildtype (JFH1) and mutant (JFH1-M) HCV.

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Construct, Luciferase, Activity Assay, Transfection, Mutagenesis

    miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).

    Journal: eLife

    Article Title: MicroRNA-122 supports robust innate immunity in hepatocytes by targeting the RTKs/STAT3 signaling pathway

    doi: 10.7554/eLife.41159

    Figure Lengend Snippet: miR-122 regulates IFN response by repressing STAT3 phosphorylation. ( A ) Analysis of p-STAT1 expression in HepG2 cells first transfected with mimics (NC or miR-122) for 2 days and then treated with IFN-β or IL-29 for 5–60 min. ( B ) qRT-PCR analysis of the five SOCS genes in HepG2 cells first treated with mimics for 2 days, and then transfected with JFH1 RNA for 24 hr. ( C ) qRT-PCR analysis of STAT3 mRNA in HepG2 cells, treated as in panel B. ( D ) Analysis of total and phosphorylated STAT3 in HepG2 cells treated with three independent siRNAs (si-1, si-2 and si-3) at a final concentration of 20 nM. ( E ) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with STAT3 siRNA and then treated with JFH1 RNA or poly(I:C). ( F ) Analysis of the dose-dependent effects of cryptotanshinone (CTS) and S3I-201 on p-STAT3. HepG2 cells were treated with either CST or S3I-201 at the indicated concentrations for 24 hr. qRT-PCR data are from one experiment that was representative of two ( B ) or three ( C ) independent experiments (mean ± SEM of technical triplicates).

    Article Snippet: RT reactions for HCV RNA were performed using SuperScript III Reverse Transcriptase, with a specific primer. qPCRs were performed with GoTaq qPCR Master Mix (Promega, A6002).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Concentration Assay