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GE Healthcare total ribonucleic acid rna
Total Ribonucleic Acid Rna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/total ribonucleic acid rna/product/GE Healthcare
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
total ribonucleic acid rna - by Bioz Stars, 2020-04
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Related Articles

MTT Assay:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: The dosages of AA in this study were of a non-cytotoxic dose and this was determined based on the results of the MTT assay. .. Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Synthesized:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: .. Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions. .. The Reverse transcriptase − quantitative PCR (RT-qPCR) was carried out using a SensiFAST™ SYBR® No-ROX Kit (Bioline, USA) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, United States).

Article Title: Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells
Article Snippet: Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). .. Complementary DNA (cDNA) was synthesized using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Isolation:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: .. Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions. .. The Reverse transcriptase − quantitative PCR (RT-qPCR) was carried out using a SensiFAST™ SYBR® No-ROX Kit (Bioline, USA) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, United States).

Article Title: Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice
Article Snippet: .. Total ribonucleic acid (RNA) (i.e. 3 μg) was isolated from liver tissue using the RNAspin mini RNA isolation kit (GE HealthCare, USA) and was reverse transcribed to copy DNA using random primers and VILO Master Mix kit (Invitrogen). .. Collagen α1(I) primers and probes (assay ID Mm00801666_g1) for real-time PCR were purchased from Applied Biosystems (USA).

Article Title: Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells
Article Snippet: .. Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). .. Complementary DNA (cDNA) was synthesized using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Quantitative RT-PCR:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions. .. The Reverse transcriptase − quantitative PCR (RT-qPCR) was carried out using a SensiFAST™ SYBR® No-ROX Kit (Bioline, USA) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, United States).

Article Title: Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells
Article Snippet: .. Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). .. Complementary DNA (cDNA) was synthesized using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Real-time Polymerase Chain Reaction:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions. .. The Reverse transcriptase − quantitative PCR (RT-qPCR) was carried out using a SensiFAST™ SYBR® No-ROX Kit (Bioline, USA) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, United States).

Article Title: Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice
Article Snippet: Paragraph title: 2.9. Quantitative real-time PCR analysis ... Total ribonucleic acid (RNA) (i.e. 3 μg) was isolated from liver tissue using the RNAspin mini RNA isolation kit (GE HealthCare, USA) and was reverse transcribed to copy DNA using random primers and VILO Master Mix kit (Invitrogen).

Article Title: Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells
Article Snippet: Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). .. The RT-qPCR was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad, Singapore) on a Chromo4™ Real-Time PCR Detector (Bio-Rad, USA) with gene specific primers ( ).

Incubation:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: 2.7 Cardiomyogenic specific genes expression analysis The hAF cells (3 samples) were cultivated in a 24-well culture plate at a density of 5×104 cells and were incubated at 37 °C, 5% CO2 at 95% humidity for 24 h. Afterwards, the cells were exposed to 25, 50 and 100 μg/ml of AA for 21 days and 10μM of 5-azacytidine (5-aza) for 24 h. After 24 h of 5-aza treatment, the medium was changed to basal growth medium until the 21st day. .. Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Expressing:

Article Title: Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells
Article Snippet: Paragraph title: Cardiomyogenic specific genes expression analysis ... Total ribonucleic acid (RNA) was extracted by utilizing an Illutra RNAspin Mini RNA Isolation Kit (GE Healthcare, UK), and then the complementary DNA (cDNA) was synthesized from 0.5 μg RNA using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

Polymerase Chain Reaction:

Article Title: Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells
Article Snippet: .. Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) After 7 and 21 days of cell cultivation, total ribonucleic acid (RNA) obtained from the basal and the cardiogenic induced groups (n=5) were prepared using an Illutra RNAspin Mini RNA Isolation kit (GE Healthcare, LittleChalfont, UK). .. Complementary DNA (cDNA) was synthesized using the iScript™ cDNA Synthesis kit (Bioline, USA) according to the manufacturer's instructions.

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  • 86
    GE Healthcare real time quantitative rt pcr total rna
    Efficiency of IDE down-regulation by siRNA. A , percentage of IDE mRNA inhibition, measured by <t>qRT-PCR,</t> in HeLa HHD cells transfected with different concentrations (4×100 or 4×400 nM) of specific siRNA (left panel), and at different time points after transfection by 4×100 nM siRNA (right panel). B , fluorescence microscopy analysis of HeLa HHD cells transfected with 400 nM of siIDE and siNTG 72 h after transfection. IDE expression was detected by staining with anti-IDE mAb 9B12. C , IDE expression by HeLa cells (left panel) and HEK293 cells (right panel) transfected with 4×100 nM of siIDE or siNTG and probed 48 h later by immunoblot. β 2- m served as a loading control. siNTG, small interfering <t>RNA,</t> non-targeted; siIDE, small interfering RNA, IDE-specific; β 2- m, beta 2-microglobulin. One out of 5 ( A, C ) and 2 ( B ) experiments is shown.
    Real Time Quantitative Rt Pcr Total Rna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time quantitative rt pcr total rna/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86/100 stars
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    86
    GE Healthcare reverse transcription real time pcr total cellular rna
    Effects of methanol fractions of seed shell (SM) and seed pomace (PM) on LPS-mediated iNOS and HO-1 expression. ( a ) RAW 264.7 macrophages were cultured with indicated reagent in 6-well plates for 16 h. Total cell lysates were prepared and the iNOS and HO-1 protein expression were detected by Western blotting, as described in Materials and Methods. The levels of α-tubulin in the total lysates serve as the loading control; ( b ) Band intensities were quantified by ImageJ software and indicated as relative folds of iNOS/α-tubulin and HO-1/α-tubulin. This experiment was replicated three times with similar results; ( c ) RAW 264.7 cells were cultured as described above for 12 h. Total <t>RNA</t> was prepared and the mRNA levels of iNOS were quantified by <t>RT-Q-PCR</t> relative to β-actin, as described in Materials and Methods. Data are represented as the mean ± SD ( n = 3). ** p
    Reverse Transcription Real Time Pcr Total Cellular Rna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reverse transcription real time pcr total cellular rna/product/GE Healthcare
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    91
    GE Healthcare quantitative real time polymerase chain reaction qrt pcr total rna
    Anti-TNF therapy downregulates PLD2 expression. (a) Patients with A-CD ( n = 17) were treated with anti-TNF mAb (IFX, 5 mg/kg) as indicated. Intestinal mucosal biopsies were collected before and at week 12 after the first infusion. Expression of PLD2 mRNA in intestinal mucosa was detected by <t>qRT-PCR.</t> ∗∗ p
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quantitative real time polymerase chain reaction qrt pcr total rna - by Bioz Stars, 2020-04
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    95
    GE Healthcare rna
    <t>RNA-binding</t> activity of 33N-term hAPE1. ( A ) Primary structure and biophysical parameters of 33N-term hAPE1. ( B , left) 33N-term hAPE1 shows RNA-binding ability. EMSA experiments were performed as described in ‘Materials and Methods’ section with purified recombinant hAPE1 or a synthetic 1–33 hAPE1 peptide (33N-term hAPE1). Competition experiments were performed with the indicated amount of peptide co-incubated with the hAPE1 during the binding reaction. Mixtures were separated on a native 10% (w/v) polyacrylamide gels at 120 V for 2 h. F indicates the free 34FRNA oligonucleotide 32 P-labelled probe. The image is from a representative gel out of three independent experiments, (B, right) 33N-term hAPE1 competes with hAPE1 for 28S rRNA binding. <t>GST</t> pull-down experiments were performed with total RNA extracted from HeLa cells as prey, a 10-fold molar excess of the 33N-term hAPE1 synthetic peptide as competitor and GST, GST–hAPE1 or GST-hAPE1NΔ33 as baits (see ‘Materials and Methods’ section for details). Histograms represent average values with SDs of three independent experiments.
    Rna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/GE Healthcare
    Average 95 stars, based on 202 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    Image Search Results


    Efficiency of IDE down-regulation by siRNA. A , percentage of IDE mRNA inhibition, measured by qRT-PCR, in HeLa HHD cells transfected with different concentrations (4×100 or 4×400 nM) of specific siRNA (left panel), and at different time points after transfection by 4×100 nM siRNA (right panel). B , fluorescence microscopy analysis of HeLa HHD cells transfected with 400 nM of siIDE and siNTG 72 h after transfection. IDE expression was detected by staining with anti-IDE mAb 9B12. C , IDE expression by HeLa cells (left panel) and HEK293 cells (right panel) transfected with 4×100 nM of siIDE or siNTG and probed 48 h later by immunoblot. β 2- m served as a loading control. siNTG, small interfering RNA, non-targeted; siIDE, small interfering RNA, IDE-specific; β 2- m, beta 2-microglobulin. One out of 5 ( A, C ) and 2 ( B ) experiments is shown.

    Journal: PLoS ONE

    Article Title: No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules

    doi: 10.1371/journal.pone.0088365

    Figure Lengend Snippet: Efficiency of IDE down-regulation by siRNA. A , percentage of IDE mRNA inhibition, measured by qRT-PCR, in HeLa HHD cells transfected with different concentrations (4×100 or 4×400 nM) of specific siRNA (left panel), and at different time points after transfection by 4×100 nM siRNA (right panel). B , fluorescence microscopy analysis of HeLa HHD cells transfected with 400 nM of siIDE and siNTG 72 h after transfection. IDE expression was detected by staining with anti-IDE mAb 9B12. C , IDE expression by HeLa cells (left panel) and HEK293 cells (right panel) transfected with 4×100 nM of siIDE or siNTG and probed 48 h later by immunoblot. β 2- m served as a loading control. siNTG, small interfering RNA, non-targeted; siIDE, small interfering RNA, IDE-specific; β 2- m, beta 2-microglobulin. One out of 5 ( A, C ) and 2 ( B ) experiments is shown.

    Article Snippet: RNA Isolation, cDNA Synthesis and Real-time Quantitative RT-PCR Total RNA was extracted from 2×106 siRNA-transfected cells with the QuickPrep™ extraction kit (GE Healthcare).

    Techniques: Inhibition, Quantitative RT-PCR, Transfection, Fluorescence, Microscopy, Expressing, Staining, Small Interfering RNA

    Effects of methanol fractions of seed shell (SM) and seed pomace (PM) on LPS-mediated iNOS and HO-1 expression. ( a ) RAW 264.7 macrophages were cultured with indicated reagent in 6-well plates for 16 h. Total cell lysates were prepared and the iNOS and HO-1 protein expression were detected by Western blotting, as described in Materials and Methods. The levels of α-tubulin in the total lysates serve as the loading control; ( b ) Band intensities were quantified by ImageJ software and indicated as relative folds of iNOS/α-tubulin and HO-1/α-tubulin. This experiment was replicated three times with similar results; ( c ) RAW 264.7 cells were cultured as described above for 12 h. Total RNA was prepared and the mRNA levels of iNOS were quantified by RT-Q-PCR relative to β-actin, as described in Materials and Methods. Data are represented as the mean ± SD ( n = 3). ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Antioxidant, Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria

    doi: 10.3390/ijms161226184

    Figure Lengend Snippet: Effects of methanol fractions of seed shell (SM) and seed pomace (PM) on LPS-mediated iNOS and HO-1 expression. ( a ) RAW 264.7 macrophages were cultured with indicated reagent in 6-well plates for 16 h. Total cell lysates were prepared and the iNOS and HO-1 protein expression were detected by Western blotting, as described in Materials and Methods. The levels of α-tubulin in the total lysates serve as the loading control; ( b ) Band intensities were quantified by ImageJ software and indicated as relative folds of iNOS/α-tubulin and HO-1/α-tubulin. This experiment was replicated three times with similar results; ( c ) RAW 264.7 cells were cultured as described above for 12 h. Total RNA was prepared and the mRNA levels of iNOS were quantified by RT-Q-PCR relative to β-actin, as described in Materials and Methods. Data are represented as the mean ± SD ( n = 3). ** p

    Article Snippet: RNA Extraction and Reverse Transcription Real-Time PCR Total cellular RNA was extracted from RAW 264.7 cells using Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare).

    Techniques: Expressing, Cell Culture, Western Blot, Software, Polymerase Chain Reaction

    Involvement of HO-1 up-regulation in the anti-inflammatory effect of the methanol fraction of seed shell (SM). ( a ) RAW 264.7 macrophages were cultured with indicated reagent in 6-well plates for 12 h. Total RNA was prepared and the mRNA levels of HO-1 were quantified by RT-Q-PCR relative to β-actin. Data are represented as the mean ± SD ( n = 3). ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Antioxidant, Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria

    doi: 10.3390/ijms161226184

    Figure Lengend Snippet: Involvement of HO-1 up-regulation in the anti-inflammatory effect of the methanol fraction of seed shell (SM). ( a ) RAW 264.7 macrophages were cultured with indicated reagent in 6-well plates for 12 h. Total RNA was prepared and the mRNA levels of HO-1 were quantified by RT-Q-PCR relative to β-actin. Data are represented as the mean ± SD ( n = 3). ** p

    Article Snippet: RNA Extraction and Reverse Transcription Real-Time PCR Total cellular RNA was extracted from RAW 264.7 cells using Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare).

    Techniques: Cell Culture, Polymerase Chain Reaction

    The inhibitory effects of methanol fractions of seed shell (SM) and seed pomace (PM) on LPS-induced IL-6 release and IL-1β mRNA expression. ( a ) RAW 264.7 cells were treated with indicated reagent for 18 h and the culture media were collected for IL-6 assay, as described in Materials and Methods; ( b ) RAW 264.7 macrophages were cultured with indicated reagent in six-well plates for 12 h. Total RNA was prepared and the mRNA levels of IL-1β were quantified by RT-Q-PCR relative to β-actin, as described in Materials and Methods. Data are represented as the mean ± SD ( n = 3). ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Antioxidant, Anti-Tyrosinase and Anti-Inflammatory Activities of Oil Production Residues from Camellia tenuifloria

    doi: 10.3390/ijms161226184

    Figure Lengend Snippet: The inhibitory effects of methanol fractions of seed shell (SM) and seed pomace (PM) on LPS-induced IL-6 release and IL-1β mRNA expression. ( a ) RAW 264.7 cells were treated with indicated reagent for 18 h and the culture media were collected for IL-6 assay, as described in Materials and Methods; ( b ) RAW 264.7 macrophages were cultured with indicated reagent in six-well plates for 12 h. Total RNA was prepared and the mRNA levels of IL-1β were quantified by RT-Q-PCR relative to β-actin, as described in Materials and Methods. Data are represented as the mean ± SD ( n = 3). ** p

    Article Snippet: RNA Extraction and Reverse Transcription Real-Time PCR Total cellular RNA was extracted from RAW 264.7 cells using Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare).

    Techniques: Expressing, Cell Culture, Polymerase Chain Reaction

    Anti-TNF therapy downregulates PLD2 expression. (a) Patients with A-CD ( n = 17) were treated with anti-TNF mAb (IFX, 5 mg/kg) as indicated. Intestinal mucosal biopsies were collected before and at week 12 after the first infusion. Expression of PLD2 mRNA in intestinal mucosa was detected by qRT-PCR. ∗∗ p

    Journal: Mediators of Inflammation

    Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

    doi: 10.1155/2016/2543070

    Figure Lengend Snippet: Anti-TNF therapy downregulates PLD2 expression. (a) Patients with A-CD ( n = 17) were treated with anti-TNF mAb (IFX, 5 mg/kg) as indicated. Intestinal mucosal biopsies were collected before and at week 12 after the first infusion. Expression of PLD2 mRNA in intestinal mucosa was detected by qRT-PCR. ∗∗ p

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from the fresh-frozen biopsies or mouse colonic tissues, and the quantity and quality were assessed using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA), with a 260/280 ratio between 1.8 and 2.0.

    Techniques: Expressing, Quantitative RT-PCR

    PLD2 is mainly expressed in neutrophils. (a) Expression of PLD2 in different subsets of immune cells. Peripheral neutrophils, CD20 + B cells, CD8 + T cells, CD4 + T cells, and CD14 + monocytes (1 × 10 6 ) were isolated from healthy donors ( n = 10), and expression of PLD2 was detected by qRT-PCR. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

    doi: 10.1155/2016/2543070

    Figure Lengend Snippet: PLD2 is mainly expressed in neutrophils. (a) Expression of PLD2 in different subsets of immune cells. Peripheral neutrophils, CD20 + B cells, CD8 + T cells, CD4 + T cells, and CD14 + monocytes (1 × 10 6 ) were isolated from healthy donors ( n = 10), and expression of PLD2 was detected by qRT-PCR. ∗ p

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from the fresh-frozen biopsies or mouse colonic tissues, and the quantity and quality were assessed using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA), with a 260/280 ratio between 1.8 and 2.0.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    TNF- α upregulates PLD2 expression in neutrophils. (a) Neutrophils isolated from healthy donors ( n = 10) were stimulated with IL-6 (10 ng/mL), IL-17A (10 ng/mL), TNF- α (10 ng/mL), IFN- γ (10 ng/mL), LPS (10 ng/mL), and IL-1 β (10 ng/mL), respectively, for 4 h. Expression of PLD2 mRNA was detected by qRT-PCR. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

    doi: 10.1155/2016/2543070

    Figure Lengend Snippet: TNF- α upregulates PLD2 expression in neutrophils. (a) Neutrophils isolated from healthy donors ( n = 10) were stimulated with IL-6 (10 ng/mL), IL-17A (10 ng/mL), TNF- α (10 ng/mL), IFN- γ (10 ng/mL), LPS (10 ng/mL), and IL-1 β (10 ng/mL), respectively, for 4 h. Expression of PLD2 mRNA was detected by qRT-PCR. ∗ p

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from the fresh-frozen biopsies or mouse colonic tissues, and the quantity and quality were assessed using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA), with a 260/280 ratio between 1.8 and 2.0.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    PLD2 is highly expressed in patients with active IBD. (a) Peripheral blood samples were collected from patients with active CD (A-CD, n = 25), patients with CD in remission (R-CD, n = 19), patients with active UC (A-UC, n = 20), patients with UC in remission (R-UC, n = 21), and healthy controls ( n = 28). Expression of PLD2 mRNA was detected by qRT-PCR. (b) Colonic biopsies were collected from patients with A-CD ( n = 21), R-CD ( n = 27), A-UC ( n = 26), R-UC ( n = 26), and HC ( n = 18). Expression of PLD2 mRNA was examined by qRT-PCR. Gene expression was normalized to GAPDH in each group. ∗∗ p

    Journal: Mediators of Inflammation

    Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

    doi: 10.1155/2016/2543070

    Figure Lengend Snippet: PLD2 is highly expressed in patients with active IBD. (a) Peripheral blood samples were collected from patients with active CD (A-CD, n = 25), patients with CD in remission (R-CD, n = 19), patients with active UC (A-UC, n = 20), patients with UC in remission (R-UC, n = 21), and healthy controls ( n = 28). Expression of PLD2 mRNA was detected by qRT-PCR. (b) Colonic biopsies were collected from patients with A-CD ( n = 21), R-CD ( n = 27), A-UC ( n = 26), R-UC ( n = 26), and HC ( n = 18). Expression of PLD2 mRNA was examined by qRT-PCR. Gene expression was normalized to GAPDH in each group. ∗∗ p

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from the fresh-frozen biopsies or mouse colonic tissues, and the quantity and quality were assessed using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA), with a 260/280 ratio between 1.8 and 2.0.

    Techniques: Expressing, Quantitative RT-PCR

    Cytokines profiles in colonic tissues from DSS-induced colitis mice after PLD2 inhibition. ((a)–(f)) Colonic tissues were obtained from mice on day 10 after DSS-induced colitis, and total RNA was extracted to detect mRNA levels of various cytokines by qRT-PCR. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Blockade of PLD2 Ameliorates Intestinal Mucosal Inflammation of Inflammatory Bowel Disease

    doi: 10.1155/2016/2543070

    Figure Lengend Snippet: Cytokines profiles in colonic tissues from DSS-induced colitis mice after PLD2 inhibition. ((a)–(f)) Colonic tissues were obtained from mice on day 10 after DSS-induced colitis, and total RNA was extracted to detect mRNA levels of various cytokines by qRT-PCR. ∗ p

    Article Snippet: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from the fresh-frozen biopsies or mouse colonic tissues, and the quantity and quality were assessed using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ, USA), with a 260/280 ratio between 1.8 and 2.0.

    Techniques: Mouse Assay, Inhibition, Quantitative RT-PCR

    RNA-binding activity of 33N-term hAPE1. ( A ) Primary structure and biophysical parameters of 33N-term hAPE1. ( B , left) 33N-term hAPE1 shows RNA-binding ability. EMSA experiments were performed as described in ‘Materials and Methods’ section with purified recombinant hAPE1 or a synthetic 1–33 hAPE1 peptide (33N-term hAPE1). Competition experiments were performed with the indicated amount of peptide co-incubated with the hAPE1 during the binding reaction. Mixtures were separated on a native 10% (w/v) polyacrylamide gels at 120 V for 2 h. F indicates the free 34FRNA oligonucleotide 32 P-labelled probe. The image is from a representative gel out of three independent experiments, (B, right) 33N-term hAPE1 competes with hAPE1 for 28S rRNA binding. GST pull-down experiments were performed with total RNA extracted from HeLa cells as prey, a 10-fold molar excess of the 33N-term hAPE1 synthetic peptide as competitor and GST, GST–hAPE1 or GST-hAPE1NΔ33 as baits (see ‘Materials and Methods’ section for details). Histograms represent average values with SDs of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

    doi: 10.1093/nar/gkq691

    Figure Lengend Snippet: RNA-binding activity of 33N-term hAPE1. ( A ) Primary structure and biophysical parameters of 33N-term hAPE1. ( B , left) 33N-term hAPE1 shows RNA-binding ability. EMSA experiments were performed as described in ‘Materials and Methods’ section with purified recombinant hAPE1 or a synthetic 1–33 hAPE1 peptide (33N-term hAPE1). Competition experiments were performed with the indicated amount of peptide co-incubated with the hAPE1 during the binding reaction. Mixtures were separated on a native 10% (w/v) polyacrylamide gels at 120 V for 2 h. F indicates the free 34FRNA oligonucleotide 32 P-labelled probe. The image is from a representative gel out of three independent experiments, (B, right) 33N-term hAPE1 competes with hAPE1 for 28S rRNA binding. GST pull-down experiments were performed with total RNA extracted from HeLa cells as prey, a 10-fold molar excess of the 33N-term hAPE1 synthetic peptide as competitor and GST, GST–hAPE1 or GST-hAPE1NΔ33 as baits (see ‘Materials and Methods’ section for details). Histograms represent average values with SDs of three independent experiments.

    Article Snippet: GST pull-down assay with RNA or NPM1 and co-immunoprecipitation with NPM1 in transfected cells An amount of 0.12 nmol of either GST–hAPE1 or each GST–hAPE1 mutant protein were added, together with 0.2 nmol of NPM1 protein or 10 µg of total RNA, to 10 µl of glutathione–Sepharose 4B beads (GE Healthcare).

    Techniques: RNA Binding Assay, Activity Assay, Purification, Recombinant, Incubation, Binding Assay

    Mapping of the amino acids involved in hAPE1 binding to NPM1. ( A , left) Overlay of SPR sensorgrams relative to the binding of NPM1 to immobilized hAPE1 (see ‘Materials and Methods’ section for experimental details). (A, middle) Plot of RU max from each binding versus NPM1 concentrations (micro molar); data were fitted by non-linear regression analysis. (A, right) Overlay of SPR sensorgrams relative to the same experiment carried out on immobilized hAPE1NΔ33. ( B and C ) GST pull-down experiments with different hAPE1 deletion (B) or with K to A (C) mutants as baits and a recombinant NPM1 as prey. Pull-down samples underwent western blot analysis using either an anti-GST antibody or an anti-NPM1 antibody. The NPM1 to GST signal intensity ratio for each sample is plotted in the histogram. ( D ) K24, K25, K27, K31 and K32 residues of hAPE1 are necessary for protein nucleolar localization and binding to NPM1. HeLa cells were transfected with pcDNA5.1 expression vector containing the cDNA of Flag-tagged hAPE1 or that of hAPE1NΔ33, hAPE1K3 or hAPE1K5 mutants. Confocal microscopy (left) of HeLa cells fixed and stained with antibodies against NPM1 (red) and ectopic Flag-tagged hAPE1 (green). Total cell extracts from transiently transfected HeLa cells with empty vector, hAPE1, hAPE1NΔ33, hAPE1K3 or hAPE1K5 mutants were co-immunoprecipitated. Co-immunoprecipitated material was separated onto 10% SDS–PAGE and analysed by western blotting to evaluate the levels of NPM1 binding by using the specific antibody. ( E ) hAPE1 binding to 28S rRNA is inhibited by NPM1. GST pull-down experiments with purified hAPE1 were carried out using 100 pmol of each bait protein incubated with the indicated amount of total RNA from HeLa cells. Pull-down samples were analysed as described in Figure 2 B, The amounts of 28S rRNA recovered after pull-downs were then normalized versus the amount of bait proteins. The resulting histogram shows the average values with SD of three independent experiments. ( F ) Effect of K + concentration on the stimulation of the endonuclease activities of hAPE1 and its mutants induced by NPM1. For each recombinant hAPE1 protein, 20 pM of each enzyme were incubated with or without 0.4 nM NPM1 and reacted, in either 40 or 150 mM KCl, with the 34FDNA as described in ‘Materials and Methods’ section. Then, reaction mixtures were separated in denaturing gels, which were then scanned for quantification. Histograms, representing the fold of increase in the reaction rates of each hAPE1 recombinant proteins, are the mean of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

    doi: 10.1093/nar/gkq691

    Figure Lengend Snippet: Mapping of the amino acids involved in hAPE1 binding to NPM1. ( A , left) Overlay of SPR sensorgrams relative to the binding of NPM1 to immobilized hAPE1 (see ‘Materials and Methods’ section for experimental details). (A, middle) Plot of RU max from each binding versus NPM1 concentrations (micro molar); data were fitted by non-linear regression analysis. (A, right) Overlay of SPR sensorgrams relative to the same experiment carried out on immobilized hAPE1NΔ33. ( B and C ) GST pull-down experiments with different hAPE1 deletion (B) or with K to A (C) mutants as baits and a recombinant NPM1 as prey. Pull-down samples underwent western blot analysis using either an anti-GST antibody or an anti-NPM1 antibody. The NPM1 to GST signal intensity ratio for each sample is plotted in the histogram. ( D ) K24, K25, K27, K31 and K32 residues of hAPE1 are necessary for protein nucleolar localization and binding to NPM1. HeLa cells were transfected with pcDNA5.1 expression vector containing the cDNA of Flag-tagged hAPE1 or that of hAPE1NΔ33, hAPE1K3 or hAPE1K5 mutants. Confocal microscopy (left) of HeLa cells fixed and stained with antibodies against NPM1 (red) and ectopic Flag-tagged hAPE1 (green). Total cell extracts from transiently transfected HeLa cells with empty vector, hAPE1, hAPE1NΔ33, hAPE1K3 or hAPE1K5 mutants were co-immunoprecipitated. Co-immunoprecipitated material was separated onto 10% SDS–PAGE and analysed by western blotting to evaluate the levels of NPM1 binding by using the specific antibody. ( E ) hAPE1 binding to 28S rRNA is inhibited by NPM1. GST pull-down experiments with purified hAPE1 were carried out using 100 pmol of each bait protein incubated with the indicated amount of total RNA from HeLa cells. Pull-down samples were analysed as described in Figure 2 B, The amounts of 28S rRNA recovered after pull-downs were then normalized versus the amount of bait proteins. The resulting histogram shows the average values with SD of three independent experiments. ( F ) Effect of K + concentration on the stimulation of the endonuclease activities of hAPE1 and its mutants induced by NPM1. For each recombinant hAPE1 protein, 20 pM of each enzyme were incubated with or without 0.4 nM NPM1 and reacted, in either 40 or 150 mM KCl, with the 34FDNA as described in ‘Materials and Methods’ section. Then, reaction mixtures were separated in denaturing gels, which were then scanned for quantification. Histograms, representing the fold of increase in the reaction rates of each hAPE1 recombinant proteins, are the mean of two independent experiments.

    Article Snippet: GST pull-down assay with RNA or NPM1 and co-immunoprecipitation with NPM1 in transfected cells An amount of 0.12 nmol of either GST–hAPE1 or each GST–hAPE1 mutant protein were added, together with 0.2 nmol of NPM1 protein or 10 µg of total RNA, to 10 µl of glutathione–Sepharose 4B beads (GE Healthcare).

    Techniques: Binding Assay, SPR Assay, Recombinant, Western Blot, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, Immunoprecipitation, SDS Page, Purification, Incubation, Concentration Assay

    Lysine residues in the 23–33 basic region are responsible for hAPE1 binding to RNA. ( A , left) Lysine to alanine mutants of hAPE1 were generated through site-directed mutagenesis. Recombinant proteins were expressed in E. coli , purified to homogeneity and used for further assays. The correct molecular mass of each recombinant protein was assessed by LC–MS analysis. Representative Coomassie-stained 10% SDS–PAGE of 1 µg of each recombinant protein is shown. (A, right) Purified hAPE1, hAPE1-K3 and hAPE1-K5 proteins were assayed for their ability to bind to 34FRNA by EMSA. A representative EMSA gel image of three independent experiments is shown. ( B ) GST pull-down experiments were performed, as described in ‘Materials and Methods’ section, with total RNA extracted from HeLa cells as prey to test the RNA-binding ability of the hAPE1-K3 and hAPE1-K5 mutant proteins in comparison with the wild-type hAPE1 and the hAPE1NΔ33. After pull-down, samples underwent either western blot analysis (using an anti-GST antibody) to confirm the amount of bait protein present in each sample, or 28S rRNA quantification. The amounts of 28S rRNA recovered after pull-downs were then normalized versus the amount of bait proteins. The resulting histogram shows the average values with SD of three independent experiments (* P

    Journal: Nucleic Acids Research

    Article Title: Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

    doi: 10.1093/nar/gkq691

    Figure Lengend Snippet: Lysine residues in the 23–33 basic region are responsible for hAPE1 binding to RNA. ( A , left) Lysine to alanine mutants of hAPE1 were generated through site-directed mutagenesis. Recombinant proteins were expressed in E. coli , purified to homogeneity and used for further assays. The correct molecular mass of each recombinant protein was assessed by LC–MS analysis. Representative Coomassie-stained 10% SDS–PAGE of 1 µg of each recombinant protein is shown. (A, right) Purified hAPE1, hAPE1-K3 and hAPE1-K5 proteins were assayed for their ability to bind to 34FRNA by EMSA. A representative EMSA gel image of three independent experiments is shown. ( B ) GST pull-down experiments were performed, as described in ‘Materials and Methods’ section, with total RNA extracted from HeLa cells as prey to test the RNA-binding ability of the hAPE1-K3 and hAPE1-K5 mutant proteins in comparison with the wild-type hAPE1 and the hAPE1NΔ33. After pull-down, samples underwent either western blot analysis (using an anti-GST antibody) to confirm the amount of bait protein present in each sample, or 28S rRNA quantification. The amounts of 28S rRNA recovered after pull-downs were then normalized versus the amount of bait proteins. The resulting histogram shows the average values with SD of three independent experiments (* P

    Article Snippet: GST pull-down assay with RNA or NPM1 and co-immunoprecipitation with NPM1 in transfected cells An amount of 0.12 nmol of either GST–hAPE1 or each GST–hAPE1 mutant protein were added, together with 0.2 nmol of NPM1 protein or 10 µg of total RNA, to 10 µl of glutathione–Sepharose 4B beads (GE Healthcare).

    Techniques: Binding Assay, Generated, Mutagenesis, Recombinant, Purification, Liquid Chromatography with Mass Spectroscopy, Staining, SDS Page, RNA Binding Assay, Western Blot

    Mapping of the RNA-binding domain of hAPE1. ( A ) Schematic representation of the hAPE1 deletion mutants used. ( B–D ) An amount of 120 pmol of each bait protein was incubated with 10 µg of total RNA purified from HeLa cells, as described in ‘Materials and Methods’ section. (B, right) After GST pull-down, samples underwent either western blot analysis using an anti-GST antibody or RNA extraction and quantification by reverse transcription and Q-PCR analysis using 28S rRNA specific primers. The amounts of 28S rRNA recovered were then normalized versus the amount of bait proteins and the resulting values are plotted in the histograms in which average values with SDs of three independent experiments are plotted (* P

    Journal: Nucleic Acids Research

    Article Title: Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

    doi: 10.1093/nar/gkq691

    Figure Lengend Snippet: Mapping of the RNA-binding domain of hAPE1. ( A ) Schematic representation of the hAPE1 deletion mutants used. ( B–D ) An amount of 120 pmol of each bait protein was incubated with 10 µg of total RNA purified from HeLa cells, as described in ‘Materials and Methods’ section. (B, right) After GST pull-down, samples underwent either western blot analysis using an anti-GST antibody or RNA extraction and quantification by reverse transcription and Q-PCR analysis using 28S rRNA specific primers. The amounts of 28S rRNA recovered were then normalized versus the amount of bait proteins and the resulting values are plotted in the histograms in which average values with SDs of three independent experiments are plotted (* P

    Article Snippet: GST pull-down assay with RNA or NPM1 and co-immunoprecipitation with NPM1 in transfected cells An amount of 0.12 nmol of either GST–hAPE1 or each GST–hAPE1 mutant protein were added, together with 0.2 nmol of NPM1 protein or 10 µg of total RNA, to 10 µl of glutathione–Sepharose 4B beads (GE Healthcare).

    Techniques: RNA Binding Assay, Incubation, Purification, Western Blot, RNA Extraction, Polymerase Chain Reaction

    Proposed mechanism of regulation of hAPE1 functions and compartmentalization through acetylation of critic lysine residues. Within cells, APE1 is present with different degrees of acetylation on residues 27, 31, 32, 35 (Ac-APE1) and may also be involved in interaction with NPM1 and/or different protein partners (indicated here as ‘X’ and ‘Y’). Genotoxic stress may shift the equilibrium between the non-acetylated and the Ac-APE1 forms toward these latter and more active form, possibly by its reduced affinity for the incised DNA product. Acetylation, in turn, may inhibit binding to RNA substrates thus, possibly, this PTM may change the equilibrium of the hAPE1 pool from the RNA-bound to the DNA-bound substrates. Moreover, acetylation may also control compartmentalization of hAPE1 within specific subnuclear structures (such as nucleoli), where the enzyme could be stored once bound to interacting partners as NPM1. Whether NPM1 may specifically participate in DNA repair inside of nucleoli, or even outside of the subnuclear condensed area, is currently unknown. In the first case, the genome containing rRNA gene may be a favoured substrate for hAPE1 in vivo . On the other hand, if the latter is the case, stimuli triggering NPM1 to get out of nucleoli may affect the cell response in terms of hAPE1 impact on genome stability. Further experiments are currently underway to address these issues.

    Journal: Nucleic Acids Research

    Article Title: Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions

    doi: 10.1093/nar/gkq691

    Figure Lengend Snippet: Proposed mechanism of regulation of hAPE1 functions and compartmentalization through acetylation of critic lysine residues. Within cells, APE1 is present with different degrees of acetylation on residues 27, 31, 32, 35 (Ac-APE1) and may also be involved in interaction with NPM1 and/or different protein partners (indicated here as ‘X’ and ‘Y’). Genotoxic stress may shift the equilibrium between the non-acetylated and the Ac-APE1 forms toward these latter and more active form, possibly by its reduced affinity for the incised DNA product. Acetylation, in turn, may inhibit binding to RNA substrates thus, possibly, this PTM may change the equilibrium of the hAPE1 pool from the RNA-bound to the DNA-bound substrates. Moreover, acetylation may also control compartmentalization of hAPE1 within specific subnuclear structures (such as nucleoli), where the enzyme could be stored once bound to interacting partners as NPM1. Whether NPM1 may specifically participate in DNA repair inside of nucleoli, or even outside of the subnuclear condensed area, is currently unknown. In the first case, the genome containing rRNA gene may be a favoured substrate for hAPE1 in vivo . On the other hand, if the latter is the case, stimuli triggering NPM1 to get out of nucleoli may affect the cell response in terms of hAPE1 impact on genome stability. Further experiments are currently underway to address these issues.

    Article Snippet: GST pull-down assay with RNA or NPM1 and co-immunoprecipitation with NPM1 in transfected cells An amount of 0.12 nmol of either GST–hAPE1 or each GST–hAPE1 mutant protein were added, together with 0.2 nmol of NPM1 protein or 10 µg of total RNA, to 10 µl of glutathione–Sepharose 4B beads (GE Healthcare).

    Techniques: Binding Assay, In Vivo