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Bio-Rad polymerase chain reaction pcr machine
Polymerase Chain Reaction Pcr Machine, supplied by Bio-Rad, 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/polymerase chain reaction pcr machine/product/Bio-Rad
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
polymerase chain reaction pcr machine - by Bioz Stars, 2020-04
94/100 stars

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High Performance Liquid Chromatography:

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: The ODNs were synthesized and purified by high-performance liquid chromatography (Sangon) with sequences as follows: the STAT5 decoy ODN, 5′-AGAT TTCTAGGAA TTCAAATC-3′ (the STAT5 consensus sequence is underlined); and mutant decoy ODN, 5′-AGAT AGTAGTGTA TTCAAATC-3′ (bases matching the STAT5 consensus sequence are underlined). .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad).

Nucleic Acid Electrophoresis:

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: .. Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton. ..

Amplification:

Article Title: Comparison of Th1/Th2 cytokine profiles between primary and secondary haemophagocytic lymphohistiocytosis
Article Snippet: .. The coding regions and flanking intronic sequences of PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes, and the deep intronic sequences of intron 1 in UNC13D gene were amplified by a polymerase chain reaction (PCR) machine (BIO-RAD) in our laboratory, and the PCR products were sequenced by a DNA sequencer in Invitrogen Company (Shanghai, China). ..

Agarose Gel Electrophoresis:

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton. .. The media materials used in this study were sterile aquadest, agar (OXOID Ltd., Basingstoke, Hampshire, England), liquid De Man, Rogosa, and Sharpe (MRS) Broth (OXOID LTD., Basingstoke, Hampshire, England), nutrient broth (OXOID LTD., Basingstoke, England), 96% alcohol, gentian violet (Merck Darmstadt, German), lugol (Merck Darmstadt, German), safranin (Merck Darmstadt, German), Presto™ Mini gDNA Bacteria KIT (Biotech Ltd. New Taipei City, Taiwan), ethanol, ddH2 O, PCR reaction buffer, DNA Polymerase (Bioline), dNTPs, MgCl2 , DNase-free water, primers (forward and reverse), agarose, Gel red (Fermentas), parafilm, dye loading, DNA samples, 1 kb DNA marker (Fermentas), and dry ice.

Synthesized:

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: The ODNs were synthesized and purified by high-performance liquid chromatography (Sangon) with sequences as follows: the STAT5 decoy ODN, 5′-AGAT TTCTAGGAA TTCAAATC-3′ (the STAT5 consensus sequence is underlined); and mutant decoy ODN, 5′-AGAT AGTAGTGTA TTCAAATC-3′ (bases matching the STAT5 consensus sequence are underlined). .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad).

Mutagenesis:

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: The ODNs were synthesized and purified by high-performance liquid chromatography (Sangon) with sequences as follows: the STAT5 decoy ODN, 5′-AGAT TTCTAGGAA TTCAAATC-3′ (the STAT5 consensus sequence is underlined); and mutant decoy ODN, 5′-AGAT AGTAGTGTA TTCAAATC-3′ (bases matching the STAT5 consensus sequence are underlined). .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad).

Isolation:

Article Title: Comparison of Th1/Th2 cytokine profiles between primary and secondary haemophagocytic lymphohistiocytosis
Article Snippet: Genetic analysis Genomic DNA was isolated from peripheral blood using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). .. The coding regions and flanking intronic sequences of PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes, and the deep intronic sequences of intron 1 in UNC13D gene were amplified by a polymerase chain reaction (PCR) machine (BIO-RAD) in our laboratory, and the PCR products were sequenced by a DNA sequencer in Invitrogen Company (Shanghai, China).

Polymerase Chain Reaction:

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: .. Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: .. Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton. ..

Article Title: Comparison of Th1/Th2 cytokine profiles between primary and secondary haemophagocytic lymphohistiocytosis
Article Snippet: .. The coding regions and flanking intronic sequences of PRF1, UNC13D, STX11, STXBP2, SH2D1A, XIAP, and ITK genes, and the deep intronic sequences of intron 1 in UNC13D gene were amplified by a polymerase chain reaction (PCR) machine (BIO-RAD) in our laboratory, and the PCR products were sequenced by a DNA sequencer in Invitrogen Company (Shanghai, China). ..

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad). ..

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: .. Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Microscopy:

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: The tools used in this study were as follows: Petri dishes (Normax Marinha Grande-Portugal), Erlenmeyer flask (Phyrex, New York, USA), test tubes (Phyrex New York, USA), test tube racks, object glasses, measuring cups (Phyrex New York, USA), Beaker glasses (Phyrex, New York, USA), microtubes, analytical scales (A & D), incubator (Memmert, Schwabach, Germany), binocular microscope (Kruess, German), autoclave (Labocon, Leicester, United Kingdom), centrifuge (Eppendorf, Hamburg, Germany), oven (Memmert, Schwabach, Germany), laminar airflow (Esco Micro Pte. .. Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton.

Sampling:

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: Orangutans, who roam freely in this forest, live naturally without human intervention, in health management, food, vaccination, and the provision of worm medicine; fecal orangutan sampling was carried out in wild forest areas. .. Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton.

Marker:

Article Title: Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)
Article Snippet: Ltd, Upper Changi Singapore), colony counter, micropipette (Eppendorf, Hamburg, Germany), polymerase chain reaction (PCR) machine (BioRad, California, USA), DNA electrophoresis apparatus (BioRad, California, USA), Ultraviolet transilluminator, Gel Doc (BioRad, California, USA), Vortex (Eppendorf), hot plate (Torey Swedesboro, USA), refrigerator (Samsung), glove, power supply (BioRad California, USA), chamber and tray (BioRad, California, USA), aluminum foil, microwave oven (Panasonic, Jakarta, Indonesia label paper, tissue, and cotton. .. The media materials used in this study were sterile aquadest, agar (OXOID Ltd., Basingstoke, Hampshire, England), liquid De Man, Rogosa, and Sharpe (MRS) Broth (OXOID LTD., Basingstoke, Hampshire, England), nutrient broth (OXOID LTD., Basingstoke, England), 96% alcohol, gentian violet (Merck Darmstadt, German), lugol (Merck Darmstadt, German), safranin (Merck Darmstadt, German), Presto™ Mini gDNA Bacteria KIT (Biotech Ltd. New Taipei City, Taiwan), ethanol, ddH2 O, PCR reaction buffer, DNA Polymerase (Bioline), dNTPs, MgCl2 , DNase-free water, primers (forward and reverse), agarose, Gel red (Fermentas), parafilm, dye loading, DNA samples, 1 kb DNA marker (Fermentas), and dry ice.

Coagulation:

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: .. Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: .. Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Sequencing:

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: The ODNs were synthesized and purified by high-performance liquid chromatography (Sangon) with sequences as follows: the STAT5 decoy ODN, 5′-AGAT TTCTAGGAA TTCAAATC-3′ (the STAT5 consensus sequence is underlined); and mutant decoy ODN, 5′-AGAT AGTAGTGTA TTCAAATC-3′ (bases matching the STAT5 consensus sequence are underlined). .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad).

Magnetic Resonance Imaging:

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Article Title: A simple method for determining the coagulation threshold temperature of transparent tissue-mimicking thermal therapy gel phantoms: Validated by magnetic resonance imaging thermometry
Article Snippet: Using the programmable heating settings of a polymerase chain reaction (PCR) machine, the authors heated 50 μ l gel samples to various temperatures for 3 min and then imaged them using the BioRad Gel Doc system to determine the coagulation temperature using an opacity quantification method. .. The estimated coagulation temperatures were then validated for gel phantoms prepared with different p H levels using high-intensity focused ultrasound (HIFU) heating and magnetic resonance imaging (MRI) thermometry methods on a clinical MR-HIFU system.

Purification:

Article Title: Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth
Article Snippet: The ODNs were synthesized and purified by high-performance liquid chromatography (Sangon) with sequences as follows: the STAT5 decoy ODN, 5′-AGAT TTCTAGGAA TTCAAATC-3′ (the STAT5 consensus sequence is underlined); and mutant decoy ODN, 5′-AGAT AGTAGTGTA TTCAAATC-3′ (bases matching the STAT5 consensus sequence are underlined). .. ODNs were dissolved in a sterile annealing buffer (10 mM Tris [pH 8.0], 50 mM NaCl, 1 mM ethylenediamine tetraacetic acid) and then annealed by heating to 95°C, followed by cooling to 25°C at 5°C increments every 15 min in a polymerase chain reaction (PCR) machine (Bio-Rad).

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  • 99
    Bio-Rad cfx96 real time pcr machine
    Protein thermal shift induced by M36 binding p32. Human recombinant protein p32 was incubated with 100 µM M36 or DMSO control and protein thermal shift determined with BioRad <t>CFX96</t> real-time <t>PCR</t>
    Cfx96 Real Time Pcr Machine, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cfx96 real time pcr machine/product/Bio-Rad
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    cfx96 real time pcr machine - by Bioz Stars, 2020-04
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      Buy from Supplier

    86
    Bio-Rad myiq qrt pcr machine
    The NMP transgene is properly expressed in the rd7 retina. A. <t>RT-PCR</t> was used to detect the presence of the NMP transgene at P30. B. <t>qRT-PCR</t> was used to measure the levels of total RDS also at P30 in eyes of the indicated genotypes. Data shown here are means±SEM from 3 different eyes/group. C. Paraffin-embedded retinal sections were labeled with mAb 3B6 (red) which specifically recognizes NMP protein but not endogenous RDS. OS: outer segment, ONL: outer nuclear layer, INL: inner nuclear layer, R: rosette. Scale bar, 10 µm.
    Myiq Qrt Pcr Machine, supplied by Bio-Rad, 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/myiq qrt pcr machine/product/Bio-Rad
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    myiq qrt pcr machine - by Bioz Stars, 2020-04
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    88
    Bio-Rad cfx 384 real time system qrt pcr machine
    Depletion of OTUB1 represses TGFβ-induced transcription. ( a ) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. ( b ) C2C12 cells stably expressing a SMAD3-dependent TGFβ-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1# 1. Cells were treated with or without 50 pM TGFβ for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. ( n =3). ( c ) HaCaT cells stably expressing shRNA against OTUB1 or transfected with control (−) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. ( d ) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by <t>qRT-PCR.</t> Data are represented as mean and error bars indicate s.d. ( n =6). ( e ) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFβ for 1 h. TGFβ was then washed off and SB505124 (1 μM) added. RNA was isolated 45 min after TGFβ removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P
    Cfx 384 Real Time System Qrt Pcr Machine, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cfx 384 real time system qrt pcr machine/product/Bio-Rad
    Average 88 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Protein thermal shift induced by M36 binding p32. Human recombinant protein p32 was incubated with 100 µM M36 or DMSO control and protein thermal shift determined with BioRad CFX96 real-time PCR

    Journal: Journal of Translational Medicine

    Article Title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma

    doi: 10.1186/s12967-017-1312-7

    Figure Lengend Snippet: Protein thermal shift induced by M36 binding p32. Human recombinant protein p32 was incubated with 100 µM M36 or DMSO control and protein thermal shift determined with BioRad CFX96 real-time PCR

    Article Snippet: The thermal shift reaction was performed with a BioRad CFX96 real-time PCR machine, and analysis for binding induced shifts in thermal transition was performed with Precision Melt Analysis Software provided by the manufacturer (BioRad).

    Techniques: Binding Assay, Recombinant, Incubation, Real-time Polymerase Chain Reaction

    Amount of E. amylovora Ea110 cells on detached apple stigmas after treatment with anti- acpP -CPP1 and streptomycin. E. amylovora Ea110 cells (10 7 CFU/ml) were inoculated on the stigma of freshly opened ‘McIntosh’ flowers (1 μl per flower by a pipette). Twenty microliters of anti- acpP -CPP1 (100, 20, and 4 μM) were evenly applied onto the stigmas of each flower 2 h before and 19 h after the inoculation. Water and streptomycin (100 μM) were used as negative and positive controls. In the PNA–bacteria mix treatment, anti- acpP -CPP1 (20 μM) was mixed with an equal volume of E. amylovora cells (10 7 CFU/ml) in a microcentrifuge tube and incubated for 30 min at room temperature (22–25°C). Two microliters of the mixture were applied evenly onto the five stigmas of the flowers. All flowers were incubated at room temperature and the pathogen population on the apple stigmas was quantified at 42 h post inoculation, using a Taqman probe realtime PCR assay. Statistical analysis was performed using ANOVA with α = 0.05. The level of significance (denoted by different letters) was calculated using LSD (α = 0.05).

    Journal: Frontiers in Microbiology

    Article Title: Exploration of Using Antisense Peptide Nucleic Acid (PNA)-cell Penetrating Peptide (CPP) as a Novel Bactericide against Fire Blight Pathogen Erwinia amylovora

    doi: 10.3389/fmicb.2017.00687

    Figure Lengend Snippet: Amount of E. amylovora Ea110 cells on detached apple stigmas after treatment with anti- acpP -CPP1 and streptomycin. E. amylovora Ea110 cells (10 7 CFU/ml) were inoculated on the stigma of freshly opened ‘McIntosh’ flowers (1 μl per flower by a pipette). Twenty microliters of anti- acpP -CPP1 (100, 20, and 4 μM) were evenly applied onto the stigmas of each flower 2 h before and 19 h after the inoculation. Water and streptomycin (100 μM) were used as negative and positive controls. In the PNA–bacteria mix treatment, anti- acpP -CPP1 (20 μM) was mixed with an equal volume of E. amylovora cells (10 7 CFU/ml) in a microcentrifuge tube and incubated for 30 min at room temperature (22–25°C). Two microliters of the mixture were applied evenly onto the five stigmas of the flowers. All flowers were incubated at room temperature and the pathogen population on the apple stigmas was quantified at 42 h post inoculation, using a Taqman probe realtime PCR assay. Statistical analysis was performed using ANOVA with α = 0.05. The level of significance (denoted by different letters) was calculated using LSD (α = 0.05).

    Article Snippet: The reaction was performed on a Biorad CFX96 Realtime PCR machine.

    Techniques: Transferring, Incubation, Polymerase Chain Reaction

    The NMP transgene is properly expressed in the rd7 retina. A. RT-PCR was used to detect the presence of the NMP transgene at P30. B. qRT-PCR was used to measure the levels of total RDS also at P30 in eyes of the indicated genotypes. Data shown here are means±SEM from 3 different eyes/group. C. Paraffin-embedded retinal sections were labeled with mAb 3B6 (red) which specifically recognizes NMP protein but not endogenous RDS. OS: outer segment, ONL: outer nuclear layer, INL: inner nuclear layer, R: rosette. Scale bar, 10 µm.

    Journal: PLoS ONE

    Article Title: Overexpression of Retinal Degeneration Slow (RDS) Protein Adversely Affects Rods in the rd7 Model of Enhanced S-Cone Syndrome

    doi: 10.1371/journal.pone.0063321

    Figure Lengend Snippet: The NMP transgene is properly expressed in the rd7 retina. A. RT-PCR was used to detect the presence of the NMP transgene at P30. B. qRT-PCR was used to measure the levels of total RDS also at P30 in eyes of the indicated genotypes. Data shown here are means±SEM from 3 different eyes/group. C. Paraffin-embedded retinal sections were labeled with mAb 3B6 (red) which specifically recognizes NMP protein but not endogenous RDS. OS: outer segment, ONL: outer nuclear layer, INL: inner nuclear layer, R: rosette. Scale bar, 10 µm.

    Article Snippet: RNase-free DNase treatment was performed with DNase (Life Technologies) to remove genomic DNA. cDNA synthesis by reverse transcription was performed and 2 µg of cDNA from each sample was used for qPCR. qRT-PCR was performed in triplicate on each cDNA sample with a MyIQ qRT-PCR machine (Bio-Rad) and cT values were calculated against the housekeeping gene beta-actin .

    Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Labeling

    Depletion of OTUB1 represses TGFβ-induced transcription. ( a ) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. ( b ) C2C12 cells stably expressing a SMAD3-dependent TGFβ-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1# 1. Cells were treated with or without 50 pM TGFβ for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. ( n =3). ( c ) HaCaT cells stably expressing shRNA against OTUB1 or transfected with control (−) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. ( d ) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). ( e ) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFβ for 1 h. TGFβ was then washed off and SB505124 (1 μM) added. RNA was isolated 45 min after TGFβ removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

    Journal: Nature Communications

    Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3

    doi: 10.1038/ncomms3519

    Figure Lengend Snippet: Depletion of OTUB1 represses TGFβ-induced transcription. ( a ) C2C12 cells were transfected with three different siRNAs (#1, #2 and #3) targeting mouse OTUB1 (300 pM/10-cm dish each) and lysed 48 h after transfection. Extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and tubulin antibodies. ( b ) C2C12 cells stably expressing a SMAD3-dependent TGFβ-responsive CAGA-luciferase reporter construct were transfected with iFoxO4 or iOTUB1# 1. Cells were treated with or without 50 pM TGFβ for 6 h before lysis and luciferase activity was measured. Data are represented as mean and error bars indicate s.d. ( n =3). ( c ) HaCaT cells stably expressing shRNA against OTUB1 or transfected with control (−) or OTUB1 siRNA (300 pM/10-cm dish each) for 48 h were lysed, and extracts were resolved by SDS-PAGE and immunoblotted with OTUB1 and GAPDH antibodies. ( d ) HaCaT cells, transfected with human OTUB1 siRNA, human FoxO4 siRNA, or stably expressing OTUB1 shRNA, were treated with 50 pM TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). ( e ) HaCaT cells depleted of human OTUB1 or FoxO4 by RNAi were treated with 50 pM TGFβ for 1 h. TGFβ was then washed off and SB505124 (1 μM) added. RNA was isolated 45 min after TGFβ removal. Relative expression levels of OTUB1 and CTGF mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

    Article Snippet: Complementary DNA was made from 1 μg of the isolated RNA using the I-Script cDNA kit (Bio-Rad) according to the manufacturer’s protocol. qRT-PCR reactions were performed in quadruplicates according to the manufacturer’s protocol in a CFX 384 Real-time System qRT-PCR machine (Bio-Rad).

    Techniques: Transfection, SDS Page, Stable Transfection, Expressing, Luciferase, Construct, Lysis, Activity Assay, shRNA, Isolation, Quantitative RT-PCR

    OTUB1 prevents SMAD3 ubiquitylation in vitro. ( a ) For in-cell polyubiquitylation of FLAG-SMAD2/3/4, vectors encoding FLAG-SMAD2/3/4 were co-transfected with HA-NEDD4L and HA-ubiquitin in HEK293 cells and treated with 50 pM TGFβ and 10 μM bortezomib for 3 h before lysis and FLAG-SMAD2/3/4 were immunoprecipitated. An in vitro DUB assay of in-cell polyubiquitylated FLAG-SMAD2/3/4 was performed with GST-OTUB1 and the indicated GST-OTUB1 mutants in DUB assay buffer for 1 h at 30 °C. The assay mix was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( b ) An in vitro ubiquitylation assay was performed with human recombinant SMAD2. SMAD2 was incubated with E1, E2, E3 and ubiquitin in ubiquitylation assay buffer for 1 h at 30 °C. Increasing concentrations of GST-OTUB1 and GST-OTUB1 C91S (8–60 ng μl −1 ) were added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) FLAG-SMAD2/3/4 IPs (from HEK293 cells expressing FLAG-SMAD2/3/4 treated with 50 pM TGFβ for 1 h before lysis) were ubiquitylated in vitro in ubiquitylation assay buffer for 1 h at 30 °C using E1, E2, E3 and ubiquitin. The indicated DUBs were added at the start of the ubiquitylation assay (time 0) and proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) HaCaT cells were stably transfected with vectors encoding siRNA-resistant silent mutations (rescue) of the indicated OTUB1 constructs. These cells were then transfected with control FoxO4 or OTUB1 siRNA for 48 h and treated with or without TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

    Journal: Nature Communications

    Article Title: OTUB1 enhances TGF? signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3

    doi: 10.1038/ncomms3519

    Figure Lengend Snippet: OTUB1 prevents SMAD3 ubiquitylation in vitro. ( a ) For in-cell polyubiquitylation of FLAG-SMAD2/3/4, vectors encoding FLAG-SMAD2/3/4 were co-transfected with HA-NEDD4L and HA-ubiquitin in HEK293 cells and treated with 50 pM TGFβ and 10 μM bortezomib for 3 h before lysis and FLAG-SMAD2/3/4 were immunoprecipitated. An in vitro DUB assay of in-cell polyubiquitylated FLAG-SMAD2/3/4 was performed with GST-OTUB1 and the indicated GST-OTUB1 mutants in DUB assay buffer for 1 h at 30 °C. The assay mix was resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( b ) An in vitro ubiquitylation assay was performed with human recombinant SMAD2. SMAD2 was incubated with E1, E2, E3 and ubiquitin in ubiquitylation assay buffer for 1 h at 30 °C. Increasing concentrations of GST-OTUB1 and GST-OTUB1 C91S (8–60 ng μl −1 ) were added at the start of the ubiquitylation assay (time 0). Proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( c ) FLAG-SMAD2/3/4 IPs (from HEK293 cells expressing FLAG-SMAD2/3/4 treated with 50 pM TGFβ for 1 h before lysis) were ubiquitylated in vitro in ubiquitylation assay buffer for 1 h at 30 °C using E1, E2, E3 and ubiquitin. The indicated DUBs were added at the start of the ubiquitylation assay (time 0) and proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. ( d ) HaCaT cells were stably transfected with vectors encoding siRNA-resistant silent mutations (rescue) of the indicated OTUB1 constructs. These cells were then transfected with control FoxO4 or OTUB1 siRNA for 48 h and treated with or without TGFβ for 4 h before RNA isolation. Relative expression levels of indicated mRNAs were analysed by qRT-PCR. Data are represented as mean and error bars indicate s.d. ( n =6). Statistical significance of differences between experimental groups was assessed with Student’s t -test. ** P

    Article Snippet: Complementary DNA was made from 1 μg of the isolated RNA using the I-Script cDNA kit (Bio-Rad) according to the manufacturer’s protocol. qRT-PCR reactions were performed in quadruplicates according to the manufacturer’s protocol in a CFX 384 Real-time System qRT-PCR machine (Bio-Rad).

    Techniques: In Vitro, Transfection, Lysis, Immunoprecipitation, SDS Page, Ubiquitin Assay, Recombinant, Incubation, Expressing, Stable Transfection, Construct, Isolation, Quantitative RT-PCR