gfp control vector  (Lonza)


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    Structured Review

    Lonza gfp control vector
    Mutation or SNO of C144 inhibits oxidant-induced cell death <t>HEK-293</t> cells expressing either <t>GFP-tagged</t> TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p
    Gfp Control Vector, supplied by Lonza, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp control vector/product/Lonza
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    gfp control vector - by Bioz Stars, 2020-08
    88/100 stars

    Images

    1) Product Images from "S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death"

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2014.01.010

    Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p
    Figure Legend Snippet: Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p

    Techniques Used: Mutagenesis, Expressing, Imaging, Staining

    2) Product Images from "Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart"

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111448

    GAPDH overexpression increases in SNO-Hsp60, but not SNO-DHRS2 levels. SNO-Hsp60 ( A ) and SNO-DHRS2 ( B ) levels from Hep2G cells transfected with either a control GFP plasmid or siRNA scramble, a plasmid encoding DDK-tagged GAPDH for overexpression, a GAPDH siRNA for knock-down, or a plasmid encoding DDK-tagged GAPDH C150S were assessed via SNO-RAC proteomic analysis, followed by label-free peptide quantification (n = 6).
    Figure Legend Snippet: GAPDH overexpression increases in SNO-Hsp60, but not SNO-DHRS2 levels. SNO-Hsp60 ( A ) and SNO-DHRS2 ( B ) levels from Hep2G cells transfected with either a control GFP plasmid or siRNA scramble, a plasmid encoding DDK-tagged GAPDH for overexpression, a GAPDH siRNA for knock-down, or a plasmid encoding DDK-tagged GAPDH C150S were assessed via SNO-RAC proteomic analysis, followed by label-free peptide quantification (n = 6).

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    HepG2 cell line as a model system for examining GAPDH as a trans-S-nitrosylase. ( A ) Expression of neuronal and endothelial isoforms of NO synthase in HepG2 cells. Representative western blots are shown for neuronal (top right) and endothelial (top left) NO synthase and β-actin (lower). ( B ) Mitochondrial GAPDH protein levels were assessed after the addition of purified GAPDH to isolated HepG2 mitochondria. Representative western blots for GAPDH (upper), TOM20 (center), and the α subunit of F 1 F 0 -ATPase (lower) in HepG2 mitochondria. Control: non-treated mitochondrial control; GAPDH: purified GAPDH treated mitochondria; (n = 3). ( C ) and ( D ) Hep2G cells were transfected with either a control GFP plasmid or siRNA scramble, a plasmid encoding DDK-tagged GAPDH for overexpression, a GAPDH siRNA for knock-down, or a plasmid encoding DDK-tagged GAPDH C150S for overexpression. Representative western blots are shown together with the densitometry of GAPDH normalized to β-actin for total GAPDH (upper; GAPDH, GAPDH-DDK, GAPDH C150S -DDK) and β-actin (lower; *p
    Figure Legend Snippet: HepG2 cell line as a model system for examining GAPDH as a trans-S-nitrosylase. ( A ) Expression of neuronal and endothelial isoforms of NO synthase in HepG2 cells. Representative western blots are shown for neuronal (top right) and endothelial (top left) NO synthase and β-actin (lower). ( B ) Mitochondrial GAPDH protein levels were assessed after the addition of purified GAPDH to isolated HepG2 mitochondria. Representative western blots for GAPDH (upper), TOM20 (center), and the α subunit of F 1 F 0 -ATPase (lower) in HepG2 mitochondria. Control: non-treated mitochondrial control; GAPDH: purified GAPDH treated mitochondria; (n = 3). ( C ) and ( D ) Hep2G cells were transfected with either a control GFP plasmid or siRNA scramble, a plasmid encoding DDK-tagged GAPDH for overexpression, a GAPDH siRNA for knock-down, or a plasmid encoding DDK-tagged GAPDH C150S for overexpression. Representative western blots are shown together with the densitometry of GAPDH normalized to β-actin for total GAPDH (upper; GAPDH, GAPDH-DDK, GAPDH C150S -DDK) and β-actin (lower; *p

    Techniques Used: Expressing, Western Blot, Purification, Isolation, Transfection, Plasmid Preparation, Over Expression

    3) Product Images from "S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death"

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2014.01.010

    TRIM72 WT and TRIM72 C144S translocate to the plasma membrane in the presence of cell damage and oxidant stress HEK293 cells expressing GFP-tagged TRIM72 WT or TRIM C144S were treated with saponin (0.001%) to induce membrane damage and H 2 O 2 (200 μmol/L). Cells were imaged for 5 minutes at 60× magnification using a Zeiss LSM510 Confocal Microscope.
    Figure Legend Snippet: TRIM72 WT and TRIM72 C144S translocate to the plasma membrane in the presence of cell damage and oxidant stress HEK293 cells expressing GFP-tagged TRIM72 WT or TRIM C144S were treated with saponin (0.001%) to induce membrane damage and H 2 O 2 (200 μmol/L). Cells were imaged for 5 minutes at 60× magnification using a Zeiss LSM510 Confocal Microscope.

    Techniques Used: Expressing, Microscopy

    Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p
    Figure Legend Snippet: Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p

    Techniques Used: Mutagenesis, Expressing, Imaging, Staining

    Related Articles

    Plasmid Preparation:

    Article Title: Lactic Acid Is Elevated in Idiopathic Pulmonary Fibrosis and Induces Myofibroblast Differentiation via pH-Dependent Activation of Transforming Growth Factor-?
    Article Snippet: .. A GFP control vector (Lonza, Hopkington, MA) was used for the transfections and yielded an approximate efficiency of 30%. .. A Student unpaired t test and one-way analysis of variance were used to establish statistical significance using Graph Pad Prism software.

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. Cells expressing a GFP control vector alone also showed a 40% decrease in viability after 24 hours. .. However, cells expressing TRIM72C144S showed significantly less H2 O2 -induced cell death compared to TRIM72WT or GFP-expressing cells.

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart
    Article Snippet: .. GAPDH-DDK or GAPDHC150S -DDK or a GFP control vector (pMax-GFP; Lonza, Walkersville, MD) was transfected into HepG2 cells using XtremeGene DNA HP (Roche) according to the manufacturer's instruction; cells were incubated with the vector/transfection mixture for 48 hours. .. GAPDH siRNA (100 pmol; Life Technologies) or a siRNA scramble (Silencer Select negative control 1; Life Technologies) was transfected into HepG2 cells using electroporation (Amaxa Nucleofector; Lonza) according to the manufacturer's instruction.

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. Conversely, cells expressing a GFP control vector (there is no TRIM72 in HEK-293 cells since it is a muscle-specific protein) showed approximately 20% cell death, and this effect could not be rescued by GSNO pre-treatment ( ). .. However, after 24 hours of H2 O2 treatment, cells expressing TRIM72WT showed a 40% decrease in viability compared to a minimal loss of viability in non-treated cells expressing TRIM72WT ( ).

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. TRIM72WT or TRIM72C144S or a GFP control vector (pMax-GFP, Lonza, Walkersville, MD) was transfected into HEK-293 cells using FugeneHD (Roche) according to the manufacturer’s instruction. .. Cells were incubated with the vector/transfection mixture for 48 hours in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies).

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. HEK-293 cells were transfected with GFP-tagged TRIM72WT , TRIM72C144S or a GFP control vector as described above. ..

    Transfection:

    Article Title: Lactic Acid Is Elevated in Idiopathic Pulmonary Fibrosis and Induces Myofibroblast Differentiation via pH-Dependent Activation of Transforming Growth Factor-?
    Article Snippet: .. A GFP control vector (Lonza, Hopkington, MA) was used for the transfections and yielded an approximate efficiency of 30%. .. A Student unpaired t test and one-way analysis of variance were used to establish statistical significance using Graph Pad Prism software.

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart
    Article Snippet: .. GAPDH-DDK or GAPDHC150S -DDK or a GFP control vector (pMax-GFP; Lonza, Walkersville, MD) was transfected into HepG2 cells using XtremeGene DNA HP (Roche) according to the manufacturer's instruction; cells were incubated with the vector/transfection mixture for 48 hours. .. GAPDH siRNA (100 pmol; Life Technologies) or a siRNA scramble (Silencer Select negative control 1; Life Technologies) was transfected into HepG2 cells using electroporation (Amaxa Nucleofector; Lonza) according to the manufacturer's instruction.

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. TRIM72WT or TRIM72C144S or a GFP control vector (pMax-GFP, Lonza, Walkersville, MD) was transfected into HEK-293 cells using FugeneHD (Roche) according to the manufacturer’s instruction. .. Cells were incubated with the vector/transfection mixture for 48 hours in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies).

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. HEK-293 cells were transfected with GFP-tagged TRIM72WT , TRIM72C144S or a GFP control vector as described above. ..

    Incubation:

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase Acts as a Mitochondrial Trans-S-Nitrosylase in the Heart
    Article Snippet: .. GAPDH-DDK or GAPDHC150S -DDK or a GFP control vector (pMax-GFP; Lonza, Walkersville, MD) was transfected into HepG2 cells using XtremeGene DNA HP (Roche) according to the manufacturer's instruction; cells were incubated with the vector/transfection mixture for 48 hours. .. GAPDH siRNA (100 pmol; Life Technologies) or a siRNA scramble (Silencer Select negative control 1; Life Technologies) was transfected into HepG2 cells using electroporation (Amaxa Nucleofector; Lonza) according to the manufacturer's instruction.

    Expressing:

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. Cells expressing a GFP control vector alone also showed a 40% decrease in viability after 24 hours. .. However, cells expressing TRIM72C144S showed significantly less H2 O2 -induced cell death compared to TRIM72WT or GFP-expressing cells.

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death
    Article Snippet: .. Conversely, cells expressing a GFP control vector (there is no TRIM72 in HEK-293 cells since it is a muscle-specific protein) showed approximately 20% cell death, and this effect could not be rescued by GSNO pre-treatment ( ). .. However, after 24 hours of H2 O2 treatment, cells expressing TRIM72WT showed a 40% decrease in viability compared to a minimal loss of viability in non-treated cells expressing TRIM72WT ( ).

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    Lonza green fluorescent protein gfp expression plasmid
    Excision and integration activities of Sleeping Beauty <t>transposase</t> mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the <t>GFP</t> coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P
    Green Fluorescent Protein Gfp Expression Plasmid, supplied by Lonza, 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/green fluorescent protein gfp expression plasmid/product/Lonza
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    green fluorescent protein gfp expression plasmid - by Bioz Stars, 2020-08
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    91
    Lonza gfp vector
    <t>BATF</t> silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg <t>GFP</t> Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P
    Gfp Vector, supplied by Lonza, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp vector/product/Lonza
    Average 91 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    gfp vector - by Bioz Stars, 2020-08
    91/100 stars
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    92
    Lonza sox17 1 gfp e bac
    <t>E-BAC</t> compartment analysis. 4C-seq was performed using a bait on the pBACe3.6 plasmid backbone. ( A ) Exemplary 4C-seq and repli-seq profiles for chr12 are plotted as a black or yellow histogram, respectively. Strong Interaction sites (SIs) with reads between 39 to 400 RPM, and Moderate Interaction site (MIs) with reads between 20 and 39 RPM, are shaded by red or yellow dashed vertical lines, respectively, demonstrating how the strong and moderate interaction sites were defined for B and C. The red and pink horizontal lines indicate the y-axis positions of the SI and MI cut-offs. ( B ) Boxplots demonstrate RT distribution of E-BAC Strong Interaction sites (SI, red box), Moderate Interaction sites (MI, pink box) and the rest of genome as control (R, gray box) for all three pools and Sox17-3 subclone 5 (Sox17-3 #5), <t>Sox17-1</t> clones 3 and 9 (Sox17-1 #3 and #9). ( C ) Venn diagram shows the number of shared SI or MI sites (50kb windows) in 4C-seq profiles of Sox17-3, Sox17-1 and BAC5 pool.
    Sox17 1 Gfp E Bac, supplied by Lonza, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sox17 1 gfp e bac/product/Lonza
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    Image Search Results


    Excision and integration activities of Sleeping Beauty transposase mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the GFP coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P

    Journal: Nucleic Acids Research

    Article Title: A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

    doi: 10.1093/nar/gkz1119

    Figure Lengend Snippet: Excision and integration activities of Sleeping Beauty transposase mutants. ( A ) Transposon excision assay. A genetically tagged SB transposon disrupts the GFP coding sequence maintained on a plasmid. Cells transfected with this construct do not express GFP. In the presence of SB transposase excision occurs, and in a fraction of the products the GFP coding sequence is restored, thereby leading to green fluorescence that can be quantified. ( B ) Relative excision efficiencies. Plasmids expressing transposase mutants were transiently cotransfected with a transposon-donor plasmid (pCMV(CAT)-GFP//T2Neo) into HeLa cells. The frequency of excision is indicated by GFP fluorescence intensity, determined by FACS analysis and normalized to SB100X. An inactive SB transposase (D3) was included as negative control. Data are represented as mean ± SD, n = 3 biological replicates. Differences in excision activities are significant as determined by Student's t-test for K248T P = 0.018, for all other mutants P

    Article Snippet: 50 ng of transposase plasmid [mutant SB100X transposase or wild-type SB100X (pCMV(CAT)T7-SB100X)] or green fluorescent protein (GFP) expression plasmid (pmaxGFP™; Lonza, Basel, Switzerland) were transfected.

    Techniques: Excision Assay, Sequencing, Plasmid Preparation, Transfection, Construct, Fluorescence, Expressing, FACS, Negative Control

    Reprogramming cassette removal with K248T in mouse induced pluripotent stem cells. ( A ) Transient transgenesis with an SB vector conta ining reprogramming genes and their removal with transient expression of the K248T SB transposase. Schematic representation of the SB transposon-based reprogramming vector pT2-CAG.OSKML-puΔtk ( 26 ). Individual genes in five-factor cassettes were linked by 2A self-cleaving peptide sequence and expressed from the CAG promoter. Black arrows represent SB transposon TIRs; pA, bovine growth hormone polyadenylation signal; puΔtk, PGK promoter-driven puΔtk expression cassette. The OSKML factors plus selection genes were removed from a single-copy mouse iPSC clone with the help of the SB100X transposase mutant K248T. ( B ) Linker-mediated PCR genotyping of a mouse iPSC clone before and after cassette removal. The lack of a PCR product in lane 2 of the agarose gel indicates that the reprogramming cassette was successfully removed with K248T (top gel). The quality of the genomic DNAs was verified by control PCR using GAPDH primers (bottom gel). ( C ) Phenotyping of a reprogramming factor-free mouse iPSC clone. Reprogramming factor-free mouse iPSCs express the endogenous nanog pluripotency marker (immunostaining) and GFP from the endogenous Oct4 promoter. Scale bar: 50 μm.

    Journal: Nucleic Acids Research

    Article Title: A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

    doi: 10.1093/nar/gkz1119

    Figure Lengend Snippet: Reprogramming cassette removal with K248T in mouse induced pluripotent stem cells. ( A ) Transient transgenesis with an SB vector conta ining reprogramming genes and their removal with transient expression of the K248T SB transposase. Schematic representation of the SB transposon-based reprogramming vector pT2-CAG.OSKML-puΔtk ( 26 ). Individual genes in five-factor cassettes were linked by 2A self-cleaving peptide sequence and expressed from the CAG promoter. Black arrows represent SB transposon TIRs; pA, bovine growth hormone polyadenylation signal; puΔtk, PGK promoter-driven puΔtk expression cassette. The OSKML factors plus selection genes were removed from a single-copy mouse iPSC clone with the help of the SB100X transposase mutant K248T. ( B ) Linker-mediated PCR genotyping of a mouse iPSC clone before and after cassette removal. The lack of a PCR product in lane 2 of the agarose gel indicates that the reprogramming cassette was successfully removed with K248T (top gel). The quality of the genomic DNAs was verified by control PCR using GAPDH primers (bottom gel). ( C ) Phenotyping of a reprogramming factor-free mouse iPSC clone. Reprogramming factor-free mouse iPSCs express the endogenous nanog pluripotency marker (immunostaining) and GFP from the endogenous Oct4 promoter. Scale bar: 50 μm.

    Article Snippet: 50 ng of transposase plasmid [mutant SB100X transposase or wild-type SB100X (pCMV(CAT)T7-SB100X)] or green fluorescent protein (GFP) expression plasmid (pmaxGFP™; Lonza, Basel, Switzerland) were transfected.

    Techniques: Plasmid Preparation, Expressing, Sequencing, Selection, Mutagenesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Immunostaining

    BATF silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg GFP Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P

    Journal: Frontiers in Immunology

    Article Title: BATF Potentially Mediates Negative Regulation of PD-1/PD-Ls Pathway on T Cell Functions in Mycobacterium tuberculosis Infection

    doi: 10.3389/fimmu.2019.02430

    Figure Lengend Snippet: BATF silencing enhanced cytokine secretions by PPD-specific T cells in PBMCs in patients with ATB. (A) Representative fluorescence microscopy image of transfected primary human PBMCs after 24 h of electroporation with 2 μg GFP Vector, and red arrow indicated transfected PBMCs containing GFP (× 200). (B) Transfection efficiency of primary human PBMCs 24 h post electroporation were assessed by analysis of GFP protein expression by flow cytometry. (C) Silencing of BATF by a BATF siRNA SMART pool in primary human PBMCs from ATB patients measured by real-time quantitative PCR. Expression normalized to a housekeeping gene ( GAPDH ) is presented as fold change relative to negative control siRNA. (D) PBMCs were electroporated with indicated siRNA, then cultured with M. tb -specific antigen PPD (25 μg/mL) overnight, and IL-2 or IFN-γ mRNA levels were measured by real-time quantitative PCR. The information of controls and cell viability after transfection was provided in the Methods section BATF Small Interfering RNA (siRNA) Knockdown in ATB Patients ( 11 ). Data are expressed as mean ± SEM ( n = 7). * P

    Article Snippet: Per 5 × 106 PBMCs from ATB patients were resuspended in 100 μL room-temperature balanced Nucleofector®solution (Human T Cell Nucleofector®Kit, Lonza, Germany), and added with 200 nM siRNA (ON-TARGET plus Non-targeting pool or BATF ON-TARGET plus SMART pool, GE Dharmacon, USA) or 2 μg GFP Vector (Lonza, Germany).

    Techniques: Fluorescence, Microscopy, Transfection, Electroporation, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Negative Control, Cell Culture, Small Interfering RNA

    Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p

    Journal: Journal of molecular and cellular cardiology

    Article Title: S-nitrosylation of TRIM72 at cysteine 144 is critical for protection against oxidation-induced protein degradation and cell death

    doi: 10.1016/j.yjmcc.2014.01.010

    Figure Lengend Snippet: Mutation or SNO of C144 inhibits oxidant-induced cell death HEK-293 cells expressing either GFP-tagged TRIM72 WT or TRIM72 C144S , or GFP alone were treated for (A) 3 hours or (B) 24 hours with H 2 O 2 (200 μmol/L) with or without GSNO pre-treatment (1 mmol/L, 1 hour). Cell viability was assayed by fluorescent imaging of ethidium bromide staining normalized to cell number (*p

    Article Snippet: HEK-293 cells were transfected with GFP-tagged TRIM72WT , TRIM72C144S or a GFP control vector as described above.

    Techniques: Mutagenesis, Expressing, Imaging, Staining

    E-BAC compartment analysis. 4C-seq was performed using a bait on the pBACe3.6 plasmid backbone. ( A ) Exemplary 4C-seq and repli-seq profiles for chr12 are plotted as a black or yellow histogram, respectively. Strong Interaction sites (SIs) with reads between 39 to 400 RPM, and Moderate Interaction site (MIs) with reads between 20 and 39 RPM, are shaded by red or yellow dashed vertical lines, respectively, demonstrating how the strong and moderate interaction sites were defined for B and C. The red and pink horizontal lines indicate the y-axis positions of the SI and MI cut-offs. ( B ) Boxplots demonstrate RT distribution of E-BAC Strong Interaction sites (SI, red box), Moderate Interaction sites (MI, pink box) and the rest of genome as control (R, gray box) for all three pools and Sox17-3 subclone 5 (Sox17-3 #5), Sox17-1 clones 3 and 9 (Sox17-1 #3 and #9). ( C ) Venn diagram shows the number of shared SI or MI sites (50kb windows) in 4C-seq profiles of Sox17-3, Sox17-1 and BAC5 pool.

    Journal: Nucleic Acids Research

    Article Title: Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

    doi: 10.1093/nar/gkx1265

    Figure Lengend Snippet: E-BAC compartment analysis. 4C-seq was performed using a bait on the pBACe3.6 plasmid backbone. ( A ) Exemplary 4C-seq and repli-seq profiles for chr12 are plotted as a black or yellow histogram, respectively. Strong Interaction sites (SIs) with reads between 39 to 400 RPM, and Moderate Interaction site (MIs) with reads between 20 and 39 RPM, are shaded by red or yellow dashed vertical lines, respectively, demonstrating how the strong and moderate interaction sites were defined for B and C. The red and pink horizontal lines indicate the y-axis positions of the SI and MI cut-offs. ( B ) Boxplots demonstrate RT distribution of E-BAC Strong Interaction sites (SI, red box), Moderate Interaction sites (MI, pink box) and the rest of genome as control (R, gray box) for all three pools and Sox17-3 subclone 5 (Sox17-3 #5), Sox17-1 clones 3 and 9 (Sox17-1 #3 and #9). ( C ) Venn diagram shows the number of shared SI or MI sites (50kb windows) in 4C-seq profiles of Sox17-3, Sox17-1 and BAC5 pool.

    Article Snippet: Sox17-1 GFP E-BAC was transfected into K3-EBNA1 cells by nucleofection according to manufacture's specifications (Lonza P3 Primary Cell Kit, V4XP-3032).

    Techniques: BAC Assay, Plasmid Preparation, Clone Assay

    E-BACs are maintained as extra-chromosomal units in Hela-EBNA1 cells at different copy numbers. ( A ) Gardella gel (Sox17-3 and sox17-1) or Hirt extraction (BAC5) results for E-BAC transfected HeLa-EBNA1 cells (Gardella and Hirt extraction give similar results, Supplementary Figure S3A ). The circular E-BAC control (E-BAC Circular) was prepared by lysing sox17-1 carrying E. coli embedded in an agarose block; linear E-BAC control (E-BAC linear) is Mlu1-linearized sox17-1 E-BAC, and DNA from E-BAC-free HeLa-EBNA1 (HeLa-EBNA1) served as the negative control. DNA was hybridized with a 5kb probe derived from BAC vector pBACe3.6. ( B ) Total Repli-seq read coverage (for early + late S fractions, Figure 4A ) for E-BAC regions. The numbers on each plot represent copy number estimation of each E-BAC (see Materials and Methods). Blue arrows indicate deletions in the E-BAC5 clones.

    Journal: Nucleic Acids Research

    Article Title: Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

    doi: 10.1093/nar/gkx1265

    Figure Lengend Snippet: E-BACs are maintained as extra-chromosomal units in Hela-EBNA1 cells at different copy numbers. ( A ) Gardella gel (Sox17-3 and sox17-1) or Hirt extraction (BAC5) results for E-BAC transfected HeLa-EBNA1 cells (Gardella and Hirt extraction give similar results, Supplementary Figure S3A ). The circular E-BAC control (E-BAC Circular) was prepared by lysing sox17-1 carrying E. coli embedded in an agarose block; linear E-BAC control (E-BAC linear) is Mlu1-linearized sox17-1 E-BAC, and DNA from E-BAC-free HeLa-EBNA1 (HeLa-EBNA1) served as the negative control. DNA was hybridized with a 5kb probe derived from BAC vector pBACe3.6. ( B ) Total Repli-seq read coverage (for early + late S fractions, Figure 4A ) for E-BAC regions. The numbers on each plot represent copy number estimation of each E-BAC (see Materials and Methods). Blue arrows indicate deletions in the E-BAC5 clones.

    Article Snippet: Sox17-1 GFP E-BAC was transfected into K3-EBNA1 cells by nucleofection according to manufacture's specifications (Lonza P3 Primary Cell Kit, V4XP-3032).

    Techniques: BAC Assay, Transfection, Blocking Assay, Negative Control, Derivative Assay, Plasmid Preparation, Clone Assay

    Replication timing of sox17-1 E-BAC in K3-EBNA1 hiPSCs. ( A ) Gardella gel result for Sox17-1 E-BAC transfected K3-EBNA1 pool. Lane 1: Sox17-1 E-BAC transfected HeLa-EBNA1 cells; Lane 2: K3-EBNA1 cells; Lane 3: Sox17-1 E-BAC transfected K3-EBNA1 pool. ( B ) Repli-seq read coverage for E-BAC regions. The numbers on each plot represent copy number estimation of Sox17-1 E-BAC (see Materials and Methods). K3-EBNA1 without E-BAC transfection was used as control (grey data points). ( C ) E-BAC replication timing in K3-EBNA1 cells. Log2 ratio of early to late coverage of K3-EBNA1 with sox17-1 E-BAC was plotted (red data points) as compared to K3-EBNA1 cells as control (gray data points). Vertical pink dashed lines indicate the Sox17-1 BAC corresponding position on chr8. RT of the 21 kb bacterial sequences of each E-BAC (pBAC3.6 E-BAC backbone) was plotted separately and indicated as horizontal lines next to the chromosomal regions (as in Figure 4B ).

    Journal: Nucleic Acids Research

    Article Title: Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

    doi: 10.1093/nar/gkx1265

    Figure Lengend Snippet: Replication timing of sox17-1 E-BAC in K3-EBNA1 hiPSCs. ( A ) Gardella gel result for Sox17-1 E-BAC transfected K3-EBNA1 pool. Lane 1: Sox17-1 E-BAC transfected HeLa-EBNA1 cells; Lane 2: K3-EBNA1 cells; Lane 3: Sox17-1 E-BAC transfected K3-EBNA1 pool. ( B ) Repli-seq read coverage for E-BAC regions. The numbers on each plot represent copy number estimation of Sox17-1 E-BAC (see Materials and Methods). K3-EBNA1 without E-BAC transfection was used as control (grey data points). ( C ) E-BAC replication timing in K3-EBNA1 cells. Log2 ratio of early to late coverage of K3-EBNA1 with sox17-1 E-BAC was plotted (red data points) as compared to K3-EBNA1 cells as control (gray data points). Vertical pink dashed lines indicate the Sox17-1 BAC corresponding position on chr8. RT of the 21 kb bacterial sequences of each E-BAC (pBAC3.6 E-BAC backbone) was plotted separately and indicated as horizontal lines next to the chromosomal regions (as in Figure 4B ).

    Article Snippet: Sox17-1 GFP E-BAC was transfected into K3-EBNA1 cells by nucleofection according to manufacture's specifications (Lonza P3 Primary Cell Kit, V4XP-3032).

    Techniques: BAC Assay, Transfection