scfv antibodies  (Qiagen)

 
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    QIAprep Spin Miniprep Kit
    Description:
    For purification of up to 20 μg molecular biology grade plasmid DNA Kit contents Qiagen QIAprep Spin Miniprep Kit 50 preps 1 to 100mL Culture Volume 50L Elution Volume Molecular Biology Grade Plasmid DNA Purification Silica Technology Spin Column Format Manual Processing 30 min Time Run 20g Yield Ideal for Fluorescent and Radioactive Sequencing Ligation Cloning Transformation etc For Purification of up to 20μg Molecular Biology Grade Plasmid DNA Includes 50 QIAprep Spin Columns Reagents Buffers 2mL Collection Tubes Benefits Ready to use plasmid DNA in minutes Reproducible yields of molecular biology grade plasmid DNA Single protocol for high and low copy vectors Even higher yields with the High Yield Supplementary Protocol Improved QIAprep 2 0 Spin Column GelPilot loading dye for convenient sample analysis
    Catalog Number:
    27104
    Price:
    92.8
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    QIAprep Spin Miniprep Kit
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    Structured Review

    Qiagen scfv antibodies
    QIAprep Spin Miniprep Kit
    For purification of up to 20 μg molecular biology grade plasmid DNA Kit contents Qiagen QIAprep Spin Miniprep Kit 50 preps 1 to 100mL Culture Volume 50L Elution Volume Molecular Biology Grade Plasmid DNA Purification Silica Technology Spin Column Format Manual Processing 30 min Time Run 20g Yield Ideal for Fluorescent and Radioactive Sequencing Ligation Cloning Transformation etc For Purification of up to 20μg Molecular Biology Grade Plasmid DNA Includes 50 QIAprep Spin Columns Reagents Buffers 2mL Collection Tubes Benefits Ready to use plasmid DNA in minutes Reproducible yields of molecular biology grade plasmid DNA Single protocol for high and low copy vectors Even higher yields with the High Yield Supplementary Protocol Improved QIAprep 2 0 Spin Column GelPilot loading dye for convenient sample analysis
    https://www.bioz.com/result/scfv antibodies/product/Qiagen
    Average 90 stars, based on 50408 article reviews
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    scfv antibodies - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis"

    Article Title: Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027756

    Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.
    Figure Legend Snippet: Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.

    Techniques Used: Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Flow Cytometry, Cytometry, Labeling, Multiplex Assay, Standard Deviation

    Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Clone Assay, Incubation, Labeling, Fluorescence, FACS, Standard Deviation

    Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Standard Deviation

    aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.
    Figure Legend Snippet: aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Recombinant, Luminex, Staining, Fluorescence, Binding Assay, Concentration Assay, Standard Deviation

    2) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

    Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbagrm.2018.03.007

    Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
    Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

    Techniques Used: Binding Assay, Amplification, Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    3) Product Images from "Understanding the Inguinal Sinus in Sheep (Ovis aries)—Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors"

    Article Title: Understanding the Inguinal Sinus in Sheep (Ovis aries)—Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18071516

    Qualitative PCR electrophoresis gel and dissociation curves of real-time PCR confirming estrogen receptor 1 ( ESR1 ), and 2 ESR2 , progesterone receptor ( PGR ) and prolactin receptor ( PRLR ) gene transcription in the IGS in different phase of the estrous cycle. Green arrow indicates the specific gene band. (F) follicular phase; (ML) mid luteal phase; (M) DNA marker; bp (base pairs). All primers validated for 80 nM in the real time PCR run. Single product confirmation with the single peak in the dissociation curve.
    Figure Legend Snippet: Qualitative PCR electrophoresis gel and dissociation curves of real-time PCR confirming estrogen receptor 1 ( ESR1 ), and 2 ESR2 , progesterone receptor ( PGR ) and prolactin receptor ( PRLR ) gene transcription in the IGS in different phase of the estrous cycle. Green arrow indicates the specific gene band. (F) follicular phase; (ML) mid luteal phase; (M) DNA marker; bp (base pairs). All primers validated for 80 nM in the real time PCR run. Single product confirmation with the single peak in the dissociation curve.

    Techniques Used: Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction, Marker

    Examples of laser-scanning confocal fluorescence (LSC—lens 63.03 oil) images of IGS. One can observe immunoreactivity towards ESR1, ESR2, PGR and PRLR in cells of the apocrine glands labeled with PE and stained for the different receptors (fluorescence in green). Use of To-Pro-3 iodide for nuclear counterstaining (fluorescence in red).
    Figure Legend Snippet: Examples of laser-scanning confocal fluorescence (LSC—lens 63.03 oil) images of IGS. One can observe immunoreactivity towards ESR1, ESR2, PGR and PRLR in cells of the apocrine glands labeled with PE and stained for the different receptors (fluorescence in green). Use of To-Pro-3 iodide for nuclear counterstaining (fluorescence in red).

    Techniques Used: Fluorescence, Labeling, Staining

    Flow cytometry analysis of gated cells of IGS. Examples of gated and dot plots and histograms showing the expression of ESR1, ESR2, PLRL and PGR. Shown flow cytometry data depict a positive cell expression towards the receptors under evaluation.
    Figure Legend Snippet: Flow cytometry analysis of gated cells of IGS. Examples of gated and dot plots and histograms showing the expression of ESR1, ESR2, PLRL and PGR. Shown flow cytometry data depict a positive cell expression towards the receptors under evaluation.

    Techniques Used: Flow Cytometry, Cytometry, Expressing

    4) Product Images from "Divergent lncRNA GATA3-AS1 Regulates GATA3 Transcription in T-Helper 2 Cells"

    Article Title: Divergent lncRNA GATA3-AS1 Regulates GATA3 Transcription in T-Helper 2 Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02512

    GATA3-AS1 is localized in the nucleus and expression increases during TH2 cell polarization . (A) Cellular and nuclear fractions of primary and effector TH2 populations analyzed via qPCR. Values represent ΔΔCT vs. whole cell GAPDH. VIM-AS1 lncRNA is a nuclear control, while HPRT mRNA represents a cytoplasm specific control. Statistical significance vs. relative cytoplasmic fraction was determined by Students T-test ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 is localized in the nucleus and expression increases during TH2 cell polarization . (A) Cellular and nuclear fractions of primary and effector TH2 populations analyzed via qPCR. Values represent ΔΔCT vs. whole cell GAPDH. VIM-AS1 lncRNA is a nuclear control, while HPRT mRNA represents a cytoplasm specific control. Statistical significance vs. relative cytoplasmic fraction was determined by Students T-test ( n = 3). * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    GATA3-AS1 RNA forms an R-Loop with the central intron of GATA3-AS1 . (A) Predictive R-Loop formations according to the R-Loop database. The largest and most likely R-Loop is circled, located between regions 2 and 3 of GATA3-AS1 . (B) DRIP assay completed with RNA isolation. Results are expressed as fold enrichment relative to the RNase H negative control. Chromatin was treated with RNase to remove R-Loops as an additional control, mean ± S.D. ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 RNA forms an R-Loop with the central intron of GATA3-AS1 . (A) Predictive R-Loop formations according to the R-Loop database. The largest and most likely R-Loop is circled, located between regions 2 and 3 of GATA3-AS1 . (B) DRIP assay completed with RNA isolation. Results are expressed as fold enrichment relative to the RNase H negative control. Chromatin was treated with RNase to remove R-Loops as an additional control, mean ± S.D. ( n = 3). * P

    Techniques Used: Isolation, Negative Control

    GATA3-AS1 is expressed in TH2 cells. (A) Genomic locations of human GATA3-AS1 known isoforms and GATA3 with directions of transcription in sense ( > ) and antisense orientations (
    Figure Legend Snippet: GATA3-AS1 is expressed in TH2 cells. (A) Genomic locations of human GATA3-AS1 known isoforms and GATA3 with directions of transcription in sense ( > ) and antisense orientations (

    Techniques Used:

    GATA3-AS1 and GATA3 form a necessary feed forward loop for TH2 polarization. (A) PBMCs were cultured under TH2 polarizing conditions for 2 days and transfected with a GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). GATA3-AS1, GATA3, IL4, IL5 , and IL13 transcripts were determined by qPCR on day 5 and results expressed relative to GAPDH . Statistical significance was determined using Students T -test by comparing GATA3-AS1 siRNA knockdown to scrambled control knockdown ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 and GATA3 form a necessary feed forward loop for TH2 polarization. (A) PBMCs were cultured under TH2 polarizing conditions for 2 days and transfected with a GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). GATA3-AS1, GATA3, IL4, IL5 , and IL13 transcripts were determined by qPCR on day 5 and results expressed relative to GAPDH . Statistical significance was determined using Students T -test by comparing GATA3-AS1 siRNA knockdown to scrambled control knockdown ( n = 3). * P

    Techniques Used: Cell Culture, Transfection, Real-time Polymerase Chain Reaction

    Expression of GATA3-AS1 changes chromatin marks throughout the GATA3-AS1 - GATA3 gene locus. TH2 cultures were transfected with an siRNA specific for GATA3-AS1 region 4 or a scrambled control siRNA on day 2 as described in Figure 3 . Cultures were harvested and processed for ChIP assays on day 5. Results are expressed as fraction of input, mean ± S.D. ( n = 3). * P
    Figure Legend Snippet: Expression of GATA3-AS1 changes chromatin marks throughout the GATA3-AS1 - GATA3 gene locus. TH2 cultures were transfected with an siRNA specific for GATA3-AS1 region 4 or a scrambled control siRNA on day 2 as described in Figure 3 . Cultures were harvested and processed for ChIP assays on day 5. Results are expressed as fraction of input, mean ± S.D. ( n = 3). * P

    Techniques Used: Expressing, Transfection, Chromatin Immunoprecipitation

    5) Product Images from "Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis"

    Article Title: Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027756

    Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.
    Figure Legend Snippet: Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.

    Techniques Used: Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    6) Product Images from "Ancient Antimicrobial Peptides Kill Antibiotic-Resistant Pathogens: Australian Mammals Provide New Options"

    Article Title: Ancient Antimicrobial Peptides Kill Antibiotic-Resistant Pathogens: Australian Mammals Provide New Options

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024030

    Tammar cathelicidin gene expression in the mammary gland throughout lactation and in pouch young skin. D = Day; PY = pouch young. Cath1 and Cath5 correspond to WAM1 and WAM2, respectively. (a) Tammar cathelicidin gene expression in the mammary gland throughout the 350 days of lactation. (b) Tammar cathelicidin gene expression in male D20–D120 pouch young skin and adult lactating female pouch skin. Female pouch young skin and adult non-lactating pouch skin showed similar expression patterns (not shown). Each age group is represented by three different samples and mammary gland and thymus tissues were used as positive controls (+). No template controls (–) were clean.
    Figure Legend Snippet: Tammar cathelicidin gene expression in the mammary gland throughout lactation and in pouch young skin. D = Day; PY = pouch young. Cath1 and Cath5 correspond to WAM1 and WAM2, respectively. (a) Tammar cathelicidin gene expression in the mammary gland throughout the 350 days of lactation. (b) Tammar cathelicidin gene expression in male D20–D120 pouch young skin and adult lactating female pouch skin. Female pouch young skin and adult non-lactating pouch skin showed similar expression patterns (not shown). Each age group is represented by three different samples and mammary gland and thymus tissues were used as positive controls (+). No template controls (–) were clean.

    Techniques Used: Expressing

    Phylogenetic tree demonstrating superior diversity of antimicrobial peptides in non-eutherian mammals. Tammar cathelicidin peptides. WAM1 (a) – KRGFGKKLRKRLKKFRNSIKKRLKNFNVVIPIPLPG from MaeuCath1 (Genbank EF624481.1), WAM2 (b) -KRGLWESLKRKATKLGDDIRNTLRNFKIKFPVPRQG from MaeuCath5 (Genbank EF624484.1), Ancestral WAM (*)- RRGFWKRLRRRLRRFGDRIRNRFRNFREKLPDPFPG. Platypus cathelicidin peptides PAM1 (c) – RTKRRIKLIKNGVKKVKDILKNNNIIILPGSNEK from OranCath1 [23] and PAM2 (d) – RPWAGNGSVHRYTVLSPRLKTQ from OranCath2 [23] .
    Figure Legend Snippet: Phylogenetic tree demonstrating superior diversity of antimicrobial peptides in non-eutherian mammals. Tammar cathelicidin peptides. WAM1 (a) – KRGFGKKLRKRLKKFRNSIKKRLKNFNVVIPIPLPG from MaeuCath1 (Genbank EF624481.1), WAM2 (b) -KRGLWESLKRKATKLGDDIRNTLRNFKIKFPVPRQG from MaeuCath5 (Genbank EF624484.1), Ancestral WAM (*)- RRGFWKRLRRRLRRFGDRIRNRFRNFREKLPDPFPG. Platypus cathelicidin peptides PAM1 (c) – RTKRRIKLIKNGVKKVKDILKNNNIIILPGSNEK from OranCath1 [23] and PAM2 (d) – RPWAGNGSVHRYTVLSPRLKTQ from OranCath2 [23] .

    Techniques Used:

    7) Product Images from "Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis"

    Article Title: Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027756

    Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Flow Cytometry, Cytometry, Labeling, Multiplex Assay, Standard Deviation

    Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Clone Assay, Incubation, Labeling, Fluorescence, FACS, Standard Deviation

    Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.
    Figure Legend Snippet: Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.

    Techniques Used: Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Standard Deviation

    aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.
    Figure Legend Snippet: aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Recombinant, Luminex, Staining, Fluorescence, Binding Assay, Concentration Assay, Standard Deviation

    8) Product Images from "SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair"

    Article Title: SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair

    Journal: Autophagy

    doi: 10.1080/15548627.2016.1210368

    SQSTM1-dependent proteasomal degradation of FLNA and RAD51 regulates DNA repair. (A) sqstm1 −/− MEFs, stably expressing FLAG-SQSTM1 ( sqstm1 −/− +FLAG-SQSTM1) were irradiated where indicated and 60 min later nuclear fractions were subjected to anti-FLAG IP. The interaction of FLAG-SQSTM1 with endogenous FLNA and RAD51 was detected by immunoblotting. (B) HeLa cells transfected with GFP-FLNA were irradiated, where indicated, and 60 min later nuclear fractions were subjected to anti-GFP IP. The interaction of GFP-FLNA with endogenous SQSTM1 and RAD51 was detected by immunoblotting. (C-D) sqstm1 −/− and Sqstm1 −/− +FLAG-SQSTM1 MEFs were irradiated and subjected to cellular fractionation at the time points indicated. Nuclear (C) and cytoplasmic (D) fractions were analyzed for FLNA, RAD51, SSQTM1 and LMNB1 as a loading control. Quantification of blots can be found in Fig. S4B-C. (E-G) sqstm1 −/− and sqstm1 −/− +FLAG-SQSTM1 MEFs were pre-incubated with MG132 or leptomycin B (Lepto B) for 3 h where indicated. Cells were irradiated with 1 Gy X-ray irradiation and incubated in the presence of MG132 or leptomycin B for a further 60 min. Nuclear fractions were analyzed for FLNA ((E)and F) and RAD51 ((E)and G) levels and quantified relative to LMNB1. n = 3; error bars represent SEM; NS, not significant; *, p
    Figure Legend Snippet: SQSTM1-dependent proteasomal degradation of FLNA and RAD51 regulates DNA repair. (A) sqstm1 −/− MEFs, stably expressing FLAG-SQSTM1 ( sqstm1 −/− +FLAG-SQSTM1) were irradiated where indicated and 60 min later nuclear fractions were subjected to anti-FLAG IP. The interaction of FLAG-SQSTM1 with endogenous FLNA and RAD51 was detected by immunoblotting. (B) HeLa cells transfected with GFP-FLNA were irradiated, where indicated, and 60 min later nuclear fractions were subjected to anti-GFP IP. The interaction of GFP-FLNA with endogenous SQSTM1 and RAD51 was detected by immunoblotting. (C-D) sqstm1 −/− and Sqstm1 −/− +FLAG-SQSTM1 MEFs were irradiated and subjected to cellular fractionation at the time points indicated. Nuclear (C) and cytoplasmic (D) fractions were analyzed for FLNA, RAD51, SSQTM1 and LMNB1 as a loading control. Quantification of blots can be found in Fig. S4B-C. (E-G) sqstm1 −/− and sqstm1 −/− +FLAG-SQSTM1 MEFs were pre-incubated with MG132 or leptomycin B (Lepto B) for 3 h where indicated. Cells were irradiated with 1 Gy X-ray irradiation and incubated in the presence of MG132 or leptomycin B for a further 60 min. Nuclear fractions were analyzed for FLNA ((E)and F) and RAD51 ((E)and G) levels and quantified relative to LMNB1. n = 3; error bars represent SEM; NS, not significant; *, p

    Techniques Used: Stable Transfection, Expressing, Irradiation, Transfection, Cell Fractionation, Incubation

    FLNA-dependent effect of SQSTM1 on DNA repair. (A) A representative blot of Flna siRNA in sqstm1 −/− and sqstm1 −/− +FLAG-SQSTM1 MEFs. (B-C) Quantification of the mean number of RAD51 (p
    Figure Legend Snippet: FLNA-dependent effect of SQSTM1 on DNA repair. (A) A representative blot of Flna siRNA in sqstm1 −/− and sqstm1 −/− +FLAG-SQSTM1 MEFs. (B-C) Quantification of the mean number of RAD51 (p

    Techniques Used:

    9) Product Images from "An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts"

    Article Title: An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00583

    Validation of miR-6891-5p-mediated posttranscriptional regulation of immunoglobulin heavy chain alpha 1 and 2 ( IGHA1 and IGHA2 ) transcripts . (A) COX cells were transduced with lentiviral constructs expressing either the scrambled control or antisense sequence of miR-6891-5p. Cells were harvested after 48 h of transduction, total RNA was purified, and both IGHA1 and IGHA2 expression were analyzed by qPCR (ΔΔCt, standard error bars shown, n = 3). (B) COX cells (5 × 10 8 ) were transduced with lentiviral construct expressing either the scrambled control or antisense sequence of miR-6891-5p. After 120 h, media were collected and analyzed by ELISA using IgA antibody (standard error bars shown, n = 3). (C) Predicted binding site and heteroduplex formed between the wild-type (WT) 3′UTR of IGHA2 and miR-6891-5p. The heteroduplex formed with IGHA1 is identical to that shown. (D) Predicted binding site and heteroduplex formed between the mutated (Mut) 3′UTR sequence of IGHA2 and miR-6891-5p. (E) Either the wild-type (WT) or mutated (Mut) 3′UTR sequence of IGHA2 was cloned downstream of the luciferase reporter, creating two separate constructs. The wild-type or mutant luciferase constructs alone or together with either the miR-6891-5p expression construct (miR overexpression) or the antisense miR-6891-5p expression construct (miR inhibition) were transfected into HEK293T cells. Luciferase assay was performed 24 h after transfection (standard error bars shown, n = 3). All p -values shown are calculated using a t -test.
    Figure Legend Snippet: Validation of miR-6891-5p-mediated posttranscriptional regulation of immunoglobulin heavy chain alpha 1 and 2 ( IGHA1 and IGHA2 ) transcripts . (A) COX cells were transduced with lentiviral constructs expressing either the scrambled control or antisense sequence of miR-6891-5p. Cells were harvested after 48 h of transduction, total RNA was purified, and both IGHA1 and IGHA2 expression were analyzed by qPCR (ΔΔCt, standard error bars shown, n = 3). (B) COX cells (5 × 10 8 ) were transduced with lentiviral construct expressing either the scrambled control or antisense sequence of miR-6891-5p. After 120 h, media were collected and analyzed by ELISA using IgA antibody (standard error bars shown, n = 3). (C) Predicted binding site and heteroduplex formed between the wild-type (WT) 3′UTR of IGHA2 and miR-6891-5p. The heteroduplex formed with IGHA1 is identical to that shown. (D) Predicted binding site and heteroduplex formed between the mutated (Mut) 3′UTR sequence of IGHA2 and miR-6891-5p. (E) Either the wild-type (WT) or mutated (Mut) 3′UTR sequence of IGHA2 was cloned downstream of the luciferase reporter, creating two separate constructs. The wild-type or mutant luciferase constructs alone or together with either the miR-6891-5p expression construct (miR overexpression) or the antisense miR-6891-5p expression construct (miR inhibition) were transfected into HEK293T cells. Luciferase assay was performed 24 h after transfection (standard error bars shown, n = 3). All p -values shown are calculated using a t -test.

    Techniques Used: Transduction, Construct, Expressing, Sequencing, Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Binding Assay, Clone Assay, Luciferase, Mutagenesis, Over Expression, Inhibition, Transfection

    10) Product Images from "Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens"

    Article Title: Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18030627

    Evaluation of the reproducibility of the barcoded cDNA library preparation procedure. ( A ) Unsupervised clustering of miRNA expression from two-year-old breast cancer #3, three-year-old breast cancer #4 and the 35-year-old benign breast tissue specimens (200, 100 and 50 ng total RNA in duplicates for each specimen); ( B ) Correlation matrices displaying reproducibility between repeats and between different RNA amounts for all three specimens; ( C ) Correlation matrices displaying reproducibility for nine FFPE RNA benign breast tissue specimens. Duplicate measures were performed within the same library (Repeat 1 (r1) and repeat 2 (r2) and between libraries (Lib#1 and Lib#2) prepared at week 1 (w1) and week 2 (w2)) for 18-, 20-, 22-, 27-, 30-, 35-year-old tissues; ( D ) Unsupervised clustering of miRNA expression data from five pairs of matched fresh or frozen and FFPE specimens and six FFPE specimens. The matched pairs include fresh and one-month-old MCF10A FFPE RNA, fresh and three-month old MCF7 FFPE RNA, frozen and four-year-old invasive breast Tumor 1 (IBC1) FFPE RNA, frozen and eight-year-old invasive breast Tumor 2 (IBC2) FFPE RNA, frozen and eight-year-old normal cervix FFPE RNA. The FFPE specimens include three distinct three-year-old normal breast FFPE RNAs (from three different individuals; F ormalin- f ixed normal br east Ff-Norm-Br#1, #2, and #3) and three distinct one-year-old invasive breast cancer FFPE specimens from three different individuals ( F ormalin- f ixed I nvasive B reast C ancer Ff-IBC#3, #4, and #5). The top 75% miRNAs detected by sequencing are represented.
    Figure Legend Snippet: Evaluation of the reproducibility of the barcoded cDNA library preparation procedure. ( A ) Unsupervised clustering of miRNA expression from two-year-old breast cancer #3, three-year-old breast cancer #4 and the 35-year-old benign breast tissue specimens (200, 100 and 50 ng total RNA in duplicates for each specimen); ( B ) Correlation matrices displaying reproducibility between repeats and between different RNA amounts for all three specimens; ( C ) Correlation matrices displaying reproducibility for nine FFPE RNA benign breast tissue specimens. Duplicate measures were performed within the same library (Repeat 1 (r1) and repeat 2 (r2) and between libraries (Lib#1 and Lib#2) prepared at week 1 (w1) and week 2 (w2)) for 18-, 20-, 22-, 27-, 30-, 35-year-old tissues; ( D ) Unsupervised clustering of miRNA expression data from five pairs of matched fresh or frozen and FFPE specimens and six FFPE specimens. The matched pairs include fresh and one-month-old MCF10A FFPE RNA, fresh and three-month old MCF7 FFPE RNA, frozen and four-year-old invasive breast Tumor 1 (IBC1) FFPE RNA, frozen and eight-year-old invasive breast Tumor 2 (IBC2) FFPE RNA, frozen and eight-year-old normal cervix FFPE RNA. The FFPE specimens include three distinct three-year-old normal breast FFPE RNAs (from three different individuals; F ormalin- f ixed normal br east Ff-Norm-Br#1, #2, and #3) and three distinct one-year-old invasive breast cancer FFPE specimens from three different individuals ( F ormalin- f ixed I nvasive B reast C ancer Ff-IBC#3, #4, and #5). The top 75% miRNAs detected by sequencing are represented.

    Techniques Used: cDNA Library Assay, Expressing, Formalin-fixed Paraffin-Embedded, Sequencing

    11) Product Images from "Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine"

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

    Journal: Nature protocols

    doi: 10.1038/nprot.2012.137

    Overview of Tet-assisted bisulfite sequencing (TAB-seq). 5-hmC is protected specifically by β-GT to generate 5-gmC, followed by oxidation of 5-mC to 5-caC by mTet1. Only 5-gmC is read as C after bisulfite treatment and PCR amplification.
    Figure Legend Snippet: Overview of Tet-assisted bisulfite sequencing (TAB-seq). 5-hmC is protected specifically by β-GT to generate 5-gmC, followed by oxidation of 5-mC to 5-caC by mTet1. Only 5-gmC is read as C after bisulfite treatment and PCR amplification.

    Techniques Used: Methylation Sequencing, Polymerase Chain Reaction, Amplification

    12) Product Images from "Stable heritable germline silencing directs somatic silencing at an endogenous locus"

    Article Title: Stable heritable germline silencing directs somatic silencing at an endogenous locus

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.01.034

    Maternally transmitted epigenetic silencing (A) Normalized sid-1 mRNA levels (relative to cpf-1 for non-normalized data). Average ± SD of at least two technical replicates. (B) RNAi sensitivity of progeny (average of 62 per worm) from (n) F2 cross progeny described in (A) and their subsequent self-progeny fed dpy-11 RNAi. (C) RNAi sensitivity of progeny (average 68 per worm) from (n) F2 sid-1+/nDf32 hemizygous or sid-1+/sid-1+ cross progeny fed dpy-11 RNAi. (D) qRT-PCR measurement of sid-1 intron 4 and intron 6 expression in single worm adults relative to cpf-1 intron 5 expression. cDNA used for qRT-PCR was generated using a gene specific primer amplifying sid-1 and cpf-1 .
    Figure Legend Snippet: Maternally transmitted epigenetic silencing (A) Normalized sid-1 mRNA levels (relative to cpf-1 for non-normalized data). Average ± SD of at least two technical replicates. (B) RNAi sensitivity of progeny (average of 62 per worm) from (n) F2 cross progeny described in (A) and their subsequent self-progeny fed dpy-11 RNAi. (C) RNAi sensitivity of progeny (average 68 per worm) from (n) F2 sid-1+/nDf32 hemizygous or sid-1+/sid-1+ cross progeny fed dpy-11 RNAi. (D) qRT-PCR measurement of sid-1 intron 4 and intron 6 expression in single worm adults relative to cpf-1 intron 5 expression. cDNA used for qRT-PCR was generated using a gene specific primer amplifying sid-1 and cpf-1 .

    Techniques Used: Quantitative RT-PCR, Expressing, Generated

    Transgenerational sid-1 silencing is associated with an increase in HRDE-1-dependent small RNAs (A–B). Frequency and distribution of sense (red) and antisense (blue) 21–26 nucleotide small RNAs over the (A) sid-1 promoter and (B) the sid-1 gene (start codon to 3′ UTR). Total read counts (upper corner) are normalized to all 21–26nt reads that map to genes. (C–D) Reads (both strands) are highly enriched for (C) 22 nucleotide RNAs with (D) a 5′ guanine in all libraries. Four Psid-1::gfp .
    Figure Legend Snippet: Transgenerational sid-1 silencing is associated with an increase in HRDE-1-dependent small RNAs (A–B). Frequency and distribution of sense (red) and antisense (blue) 21–26 nucleotide small RNAs over the (A) sid-1 promoter and (B) the sid-1 gene (start codon to 3′ UTR). Total read counts (upper corner) are normalized to all 21–26nt reads that map to genes. (C–D) Reads (both strands) are highly enriched for (C) 22 nucleotide RNAs with (D) a 5′ guanine in all libraries. Four Psid-1::gfp .

    Techniques Used:

    Genetic requirements for sid-1 silencing (A) sid-1 mRNA expression in mixed stage worms. Average (red bar) of biological replicates relative to gpd-2/3 . (B) Maintenance and initiation crosses (silencing competent hermaphrodite germline is highlighted). (C) RNAi sensitivity of progeny of (n) F2 L4 larvae produced by crosses in (B). (D) Putative piRNA 14927-1 binding site and mutant Psid-1 (piRNA-4A) sequence. (E) RNAi sensitivity of progeny of (n) F2 lines produced by injected wild-type or piRNA-4A Psid-1 DNA. N.S. = Not significant, * = p
    Figure Legend Snippet: Genetic requirements for sid-1 silencing (A) sid-1 mRNA expression in mixed stage worms. Average (red bar) of biological replicates relative to gpd-2/3 . (B) Maintenance and initiation crosses (silencing competent hermaphrodite germline is highlighted). (C) RNAi sensitivity of progeny of (n) F2 L4 larvae produced by crosses in (B). (D) Putative piRNA 14927-1 binding site and mutant Psid-1 (piRNA-4A) sequence. (E) RNAi sensitivity of progeny of (n) F2 lines produced by injected wild-type or piRNA-4A Psid-1 DNA. N.S. = Not significant, * = p

    Techniques Used: Expressing, Produced, Binding Assay, Mutagenesis, Sequencing, Injection

    Transgenerational somatic silencing (A) Progeny of wild-type or Psid-1::gfp larvae (two generations) or embryos (one generation) placed on dpy-11 RNAi food. (B–D) RNAi sensitivity of (n) worms hatched on dpy-11 RNAi food, scored as adults. To determine sid-1 genotype in (B), adults were fed act-5 RNAi (L1 arrest). (E) qRT-PCR analysis of sid-1 mRNA levels (normalized, relative to gpd-2/3 ) in young adults. Average ± SD of at least two technical replicates. (F) RNAi sensitivity of methyltransferase mutants after dpy-11 .
    Figure Legend Snippet: Transgenerational somatic silencing (A) Progeny of wild-type or Psid-1::gfp larvae (two generations) or embryos (one generation) placed on dpy-11 RNAi food. (B–D) RNAi sensitivity of (n) worms hatched on dpy-11 RNAi food, scored as adults. To determine sid-1 genotype in (B), adults were fed act-5 RNAi (L1 arrest). (E) qRT-PCR analysis of sid-1 mRNA levels (normalized, relative to gpd-2/3 ) in young adults. Average ± SD of at least two technical replicates. (F) RNAi sensitivity of methyltransferase mutants after dpy-11 .

    Techniques Used: Activated Clotting Time Assay, Quantitative RT-PCR

    Transgenerational epigenetic sid-1 silencing (A) RNAi sensitivity of > 100 progeny of L4 worms. mRNA expression in (B) mixed stage worms (biological replicate average in red), (C) single young adults (25°C, each bar represents a single worm and two technical replicates), and (D) synchronized young adults. (E) Silenced region. (F) Fraction of dpy-11 RNAi resistant F2 Psid-1 array lines (n). (G) Inherited RNAi resistance of average 103 (a, b) or 870 (c, d) progeny from 3 (a, b) or 20 (c, d) L4 larvae fed dpy-11 RNAi. (H) sid-1 expression in mixed stage line b. Expression is relative to gpd-2/3 .
    Figure Legend Snippet: Transgenerational epigenetic sid-1 silencing (A) RNAi sensitivity of > 100 progeny of L4 worms. mRNA expression in (B) mixed stage worms (biological replicate average in red), (C) single young adults (25°C, each bar represents a single worm and two technical replicates), and (D) synchronized young adults. (E) Silenced region. (F) Fraction of dpy-11 RNAi resistant F2 Psid-1 array lines (n). (G) Inherited RNAi resistance of average 103 (a, b) or 870 (c, d) progeny from 3 (a, b) or 20 (c, d) L4 larvae fed dpy-11 RNAi. (H) sid-1 expression in mixed stage line b. Expression is relative to gpd-2/3 .

    Techniques Used: Expressing

    13) Product Images from "Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors"

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207948

    Molecular structures of GABA A receptors. ( A ) A cartoon representation of the major pentameric GABA A ] by using PYMOL. ( B ) Topology of the α1 subunit. The large N-terminal domain resides in the ER lumen or extracellular space. Ala322, displayed as a space-filling model and indicated by an arrow, is located in the third transmembrane (TM3) helix. ( C ) Sequence alignment of TM3 residues of the α1, β2, β3, and γ2 subunits of GABA A receptors. The sequences are from the following Uniprot entries: GBRA1, P14867; GBRB2, P47870-1; GBRB3, P28472-1; GBRG2, P18507-2. The A322 residue in the α1 subunit is highlighted in yellow. Hydrophobic residues are conserved in this position.
    Figure Legend Snippet: Molecular structures of GABA A receptors. ( A ) A cartoon representation of the major pentameric GABA A ] by using PYMOL. ( B ) Topology of the α1 subunit. The large N-terminal domain resides in the ER lumen or extracellular space. Ala322, displayed as a space-filling model and indicated by an arrow, is located in the third transmembrane (TM3) helix. ( C ) Sequence alignment of TM3 residues of the α1, β2, β3, and γ2 subunits of GABA A receptors. The sequences are from the following Uniprot entries: GBRA1, P14867; GBRB2, P47870-1; GBRB3, P28472-1; GBRG2, P18507-2. The A322 residue in the α1 subunit is highlighted in yellow. Hydrophobic residues are conserved in this position.

    Techniques Used: Sequencing

    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Activation Assay, Expressing, Transfection, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Stable Transfection, Expressing, Cell Culture, SDS Page, Western Blot, Positive Control, Fluorescence, Incubation, Quantitation Assay

    BIX enhances the surface expression of α1 subunit variants of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels to the Na + /K + -ATPase controls is shown in ( B ) (n = 5, paired t-test). ( C ) HEK293T cells expressing α1β2γ2 receptors or α1(D219N)β2γ2 receptors were treated as in ( A ). Quantification of normalized total and surface WT α1 protein levels is shown in ( D F ) (n = 6 for total and n = 5 for surface, paired t-test). Quantification of normalized total and surface α1(D219N) protein levels is shown in ( E G ) (n = 6 for total and surface, paired t-test). ( H ) SH-SY5Y cells stably expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then surface biotinylation assay was performed as in ( A ). Quantification of normalized surface α1(A322D) protein levels is shown in ( I ) (n = 3, two tailed student t-test). * p
    Figure Legend Snippet: BIX enhances the surface expression of α1 subunit variants of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels to the Na + /K + -ATPase controls is shown in ( B ) (n = 5, paired t-test). ( C ) HEK293T cells expressing α1β2γ2 receptors or α1(D219N)β2γ2 receptors were treated as in ( A ). Quantification of normalized total and surface WT α1 protein levels is shown in ( D F ) (n = 6 for total and n = 5 for surface, paired t-test). Quantification of normalized total and surface α1(D219N) protein levels is shown in ( E G ) (n = 6 for total and surface, paired t-test). ( H ) SH-SY5Y cells stably expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then surface biotinylation assay was performed as in ( A ). Quantification of normalized surface α1(A322D) protein levels is shown in ( I ) (n = 3, two tailed student t-test). * p

    Techniques Used: Expressing, Affinity Purification, SDS Page, Western Blot, Stable Transfection, Surface Biotinylation Assay, Two Tailed Test

    14) Product Images from "Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1"

    Article Title: Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

    Journal: Scientific Reports

    doi: 10.1038/srep25802

    SLC20A2 Knock Down ablates calcitriol-mediated blockade of calcification. SaOs-2 cells were transfected with guide-RNA against SLC20A2 together with Cas-9. ( A ) Gene knockdown was confirmed by PCR amplification of the targeted region and T7 digests to confirm site cutting. Note the reduction in ~500 bp fragments and the appearance of shorter fragments in the CRISPR cells, which is the result of mismatch pairing of the DNA after repair by non-homologous end joining. ( B ) Primers were designed to flank the guide-RNA targeting site and genomic PCR performed on DNA from cells transfected with a donor plasmid (containing a Puromycin resistance gene flanked by homologous arms complimentary to the gene insertion site) with Cas-9. The reduced presence of a ~500 bp fragment is caused by the puromycin insertion, extending the size of the amplicon, thereby producing a diminished band in the PCR reaction. ( C ) Western blot analysis confirmed that protein expression was reduced in CRISPR-treated cells (representative of n = 2). ( D ) Wild type (WT) or SLC20A2 knock down (KD) SaOs-2 cells were treated for 14 days under calcifying conditions with or without calcitriol (Calc). KD cells were no longer protected from calcification by calcitriol (n = 3 independent experiments, *p
    Figure Legend Snippet: SLC20A2 Knock Down ablates calcitriol-mediated blockade of calcification. SaOs-2 cells were transfected with guide-RNA against SLC20A2 together with Cas-9. ( A ) Gene knockdown was confirmed by PCR amplification of the targeted region and T7 digests to confirm site cutting. Note the reduction in ~500 bp fragments and the appearance of shorter fragments in the CRISPR cells, which is the result of mismatch pairing of the DNA after repair by non-homologous end joining. ( B ) Primers were designed to flank the guide-RNA targeting site and genomic PCR performed on DNA from cells transfected with a donor plasmid (containing a Puromycin resistance gene flanked by homologous arms complimentary to the gene insertion site) with Cas-9. The reduced presence of a ~500 bp fragment is caused by the puromycin insertion, extending the size of the amplicon, thereby producing a diminished band in the PCR reaction. ( C ) Western blot analysis confirmed that protein expression was reduced in CRISPR-treated cells (representative of n = 2). ( D ) Wild type (WT) or SLC20A2 knock down (KD) SaOs-2 cells were treated for 14 days under calcifying conditions with or without calcitriol (Calc). KD cells were no longer protected from calcification by calcitriol (n = 3 independent experiments, *p

    Techniques Used: Transfection, Polymerase Chain Reaction, Amplification, CRISPR, Non-Homologous End Joining, Plasmid Preparation, Western Blot, Expressing

    15) Product Images from "Identification of Methylated Proteins in the Yeast Small Ribosomal Subunit: A Role for SPOUT Methyltransferases in Protein Arginine Methylation"

    Article Title: Identification of Methylated Proteins in the Yeast Small Ribosomal Subunit: A Role for SPOUT Methyltransferases in Protein Arginine Methylation

    Journal: Biochemistry

    doi: 10.1021/bi300186g

    In vitro methyltransferase activity of recombinant Yor021c protein. (A) Yor021c was expressed as a His-tagged enzyme in E. coli as described in the “Experimental Procedures” section. This protein (40 μg) was incubated with ribosomes
    Figure Legend Snippet: In vitro methyltransferase activity of recombinant Yor021c protein. (A) Yor021c was expressed as a His-tagged enzyme in E. coli as described in the “Experimental Procedures” section. This protein (40 μg) was incubated with ribosomes

    Techniques Used: In Vitro, Activity Assay, Recombinant, Incubation

    Arginine Methylation of Rps3 is Catalyzed by the Yor021c SPOUT Methyltransferase
    Figure Legend Snippet: Arginine Methylation of Rps3 is Catalyzed by the Yor021c SPOUT Methyltransferase

    Techniques Used: Methylation

    16) Product Images from "Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries"

    Article Title: Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.11.014

    Dilution of Input Capsid DNA Results in Higher Quality Multiplexed Libraries (A) Experimental design. AAV1, AAV2, AAV7, AAV8, AAV9, and Anc82 capsid sequences tagged with uniquely identifying barcodes in pSL vectors were assembled into an equimolar library and used to generate a high titer library prep. In parallel, plasmids were used to generate individual preps that were then combined into a control library. All libraries were injected into mice, and livers were harvested 7 days following injection. Barcodes were isolated from liver genomic DNA and sequenced. (B) Distribution of barcode abundances in counts per million in input plasmid library (left) multiplexed vector libraries, generated using the plasmid library (middle) and a control library of individually produced and spiked vectors (right). (C) Average of barcode abundances in liver gDNA in counts per million in mice injected with each multiplexed vector library (left) and individually produced and spiked vector library (right). n = 5 mice were injected with each library. (D) Average fold change in abundance of each barcode in liver gDNA over injected vector library. (E) Euclidian distance between the distribution of fold changes for each library.
    Figure Legend Snippet: Dilution of Input Capsid DNA Results in Higher Quality Multiplexed Libraries (A) Experimental design. AAV1, AAV2, AAV7, AAV8, AAV9, and Anc82 capsid sequences tagged with uniquely identifying barcodes in pSL vectors were assembled into an equimolar library and used to generate a high titer library prep. In parallel, plasmids were used to generate individual preps that were then combined into a control library. All libraries were injected into mice, and livers were harvested 7 days following injection. Barcodes were isolated from liver genomic DNA and sequenced. (B) Distribution of barcode abundances in counts per million in input plasmid library (left) multiplexed vector libraries, generated using the plasmid library (middle) and a control library of individually produced and spiked vectors (right). (C) Average of barcode abundances in liver gDNA in counts per million in mice injected with each multiplexed vector library (left) and individually produced and spiked vector library (right). n = 5 mice were injected with each library. (D) Average fold change in abundance of each barcode in liver gDNA over injected vector library. (E) Euclidian distance between the distribution of fold changes for each library.

    Techniques Used: Injection, Mouse Assay, Isolation, Plasmid Preparation, Generated, Produced

    17) Product Images from "An enhanced toolkit for the generation of knockout and marker-free fluorescentPlasmodium chabaudi"

    Article Title: An enhanced toolkit for the generation of knockout and marker-free fluorescentPlasmodium chabaudi

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15587.1

    Generation of Plasmo GEM resources for genetic modification of P. chabaudi. Insert-size distribution for P. chabaudi genomic DNA (gDNA) clone library showing number of clones for each genomic insert-size category, with an average genomic clone size of 6-7 kb ( A ). Size selected P. chabaudi genomic DNA fragments ( ii ) were cloned into the pJAZZ-OK Blunt vector ( i ) to generate the PcG01 and PcG02 genomic libraries. The pJAZZ-OK Blunt vector encode hairpin telomers (shown in black), a telomerase gene (TelN), replication factor and origin (repA), regulator of replication (cB) and a kanamycin resistance gene (kanR). Genomic library clones are converted into gene targeting vectors by recombineering, which introduces a dual bacterial selectable marker zeocin-pheS (zeo-pheS), ( iii ). Linear transfection-ready vectors are then generated by Gateway cloning, which facilitates the exchange of the zeo-pheS cassette for the parasite positive-negative selection marker human dihydrofolate reductase-uridyl phosphoribosyl transferase (hdhfr-yfcu), ( iv ) that are prepared for transfection by NotI digest and integrate with high efficiency into the P. chabaudi genome ( v ).
    Figure Legend Snippet: Generation of Plasmo GEM resources for genetic modification of P. chabaudi. Insert-size distribution for P. chabaudi genomic DNA (gDNA) clone library showing number of clones for each genomic insert-size category, with an average genomic clone size of 6-7 kb ( A ). Size selected P. chabaudi genomic DNA fragments ( ii ) were cloned into the pJAZZ-OK Blunt vector ( i ) to generate the PcG01 and PcG02 genomic libraries. The pJAZZ-OK Blunt vector encode hairpin telomers (shown in black), a telomerase gene (TelN), replication factor and origin (repA), regulator of replication (cB) and a kanamycin resistance gene (kanR). Genomic library clones are converted into gene targeting vectors by recombineering, which introduces a dual bacterial selectable marker zeocin-pheS (zeo-pheS), ( iii ). Linear transfection-ready vectors are then generated by Gateway cloning, which facilitates the exchange of the zeo-pheS cassette for the parasite positive-negative selection marker human dihydrofolate reductase-uridyl phosphoribosyl transferase (hdhfr-yfcu), ( iv ) that are prepared for transfection by NotI digest and integrate with high efficiency into the P. chabaudi genome ( v ).

    Techniques Used: Modification, Clone Assay, Plasmid Preparation, Marker, Transfection, Generated, Selection

    Transfection of P. chabaudi AS to create PcAS-GFP ML . A) Mature P. chabaudi AS schizonts accumulate after 4.5 hours of culture in the presence of C2 (giemsa-stained smear); infected erythrocytes were smeared 5 minutes after C2 was removed from culture. Merozoites undergoing erythrocyte invasion are arrowed. B) Schematic representation of the pCAT 230p -G6 plasmid showing hDHFR and yFcu selectable cassettes, the GFP cassette and 3’PbDHFR-TS direct repeats, allowing recombination and excision of the drug-selectable cassette. C ) Stable integration of GFP into the pc230p locus. To verify correct integration into the pc230p locus in PcAS-GFP.Δ230p DNA, 1.4kb and 1kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF x 5’intR and 3’intF x 3’intR, respectively. To verify deletion of pc230p , primers 230pF x 230pR were used to amplify a 0.37kb fragment in wild-type but not PcAS-GFP.Δ230p DNA. To verify the presence of hDHFR, primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-GFP.Δ230p but not in wild-type or PcAS-GFP ML DNA. D) Recycling of the drug-selectable cassettes. Following selection with 5-FC, recombined parasites excised the drug-selectable cassettes (insert) and an 830bp fragment was amplified from PcAS-GFP ML DNA using primers recF x recR.
    Figure Legend Snippet: Transfection of P. chabaudi AS to create PcAS-GFP ML . A) Mature P. chabaudi AS schizonts accumulate after 4.5 hours of culture in the presence of C2 (giemsa-stained smear); infected erythrocytes were smeared 5 minutes after C2 was removed from culture. Merozoites undergoing erythrocyte invasion are arrowed. B) Schematic representation of the pCAT 230p -G6 plasmid showing hDHFR and yFcu selectable cassettes, the GFP cassette and 3’PbDHFR-TS direct repeats, allowing recombination and excision of the drug-selectable cassette. C ) Stable integration of GFP into the pc230p locus. To verify correct integration into the pc230p locus in PcAS-GFP.Δ230p DNA, 1.4kb and 1kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF x 5’intR and 3’intF x 3’intR, respectively. To verify deletion of pc230p , primers 230pF x 230pR were used to amplify a 0.37kb fragment in wild-type but not PcAS-GFP.Δ230p DNA. To verify the presence of hDHFR, primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-GFP.Δ230p but not in wild-type or PcAS-GFP ML DNA. D) Recycling of the drug-selectable cassettes. Following selection with 5-FC, recombined parasites excised the drug-selectable cassettes (insert) and an 830bp fragment was amplified from PcAS-GFP ML DNA using primers recF x recR.

    Techniques Used: Transfection, Staining, Infection, Plasmid Preparation, Amplification, Selection

    Modification of non-essential loci with pCAT and pJazz plasmids. A) Left hand panel (LHP) ; schematic representation of the pCAT crmp1 -M6 plasmid and the pccrmp1 locus after transfection. Right hand panel (RHP) ; to verify correct integration into the pccrmp1 locus, 1.22kb and 1.17kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF.crmp1 x 5’intR.crmp1 and 3’intF.crmp1 x 3’intR.crmp1, respectively. To verify deletion of pccrmp1 , primers crmp1F x crmp1R were used to amplify a 306bp fragment in wild-type but not PcAS-mCh.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-mCh.Δcrmp1 but not in wt DNA. B) LHP ; schematic representation of the PGEM-600068 construct and the pccrmp1 locus after transfection. RHP ; to verify correct integration into the pccrmp1 locus, a 2.9kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp1. To verify deletion of pccrmp1 , primers crmp1F2 x crmp1R2 were used to amplify a 627bp fragment in wt but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp1 but not in wt DNA. C) Day of patency following transfecti o n with pCAT plasmids (n=6) and pJazz constructs (n=8) targeting non-essential loci. Parasitaemia was monitored by giemsa stain from days 7-16 following transfection. Patency was determined when parasitaemia was at least 0.005%. D) LHP ; schematic representation of the PGEM-610754 construct and the pccrmp4 locus after transfection into P. chabaudi AS wild-type and PcAS-GFP ml parasites. RHP ; to verify correct integration into the pccrmp4 locus, a 3.3kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp4. To verify deletion of pccrmp4 in wild-type and PcAS-GFP ml parasites, primers crmp4F x crmp4R were used to amplify a 700bp fragment in wild-type but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp4 but not in wild-type DNA.
    Figure Legend Snippet: Modification of non-essential loci with pCAT and pJazz plasmids. A) Left hand panel (LHP) ; schematic representation of the pCAT crmp1 -M6 plasmid and the pccrmp1 locus after transfection. Right hand panel (RHP) ; to verify correct integration into the pccrmp1 locus, 1.22kb and 1.17kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF.crmp1 x 5’intR.crmp1 and 3’intF.crmp1 x 3’intR.crmp1, respectively. To verify deletion of pccrmp1 , primers crmp1F x crmp1R were used to amplify a 306bp fragment in wild-type but not PcAS-mCh.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-mCh.Δcrmp1 but not in wt DNA. B) LHP ; schematic representation of the PGEM-600068 construct and the pccrmp1 locus after transfection. RHP ; to verify correct integration into the pccrmp1 locus, a 2.9kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp1. To verify deletion of pccrmp1 , primers crmp1F2 x crmp1R2 were used to amplify a 627bp fragment in wt but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp1 but not in wt DNA. C) Day of patency following transfecti o n with pCAT plasmids (n=6) and pJazz constructs (n=8) targeting non-essential loci. Parasitaemia was monitored by giemsa stain from days 7-16 following transfection. Patency was determined when parasitaemia was at least 0.005%. D) LHP ; schematic representation of the PGEM-610754 construct and the pccrmp4 locus after transfection into P. chabaudi AS wild-type and PcAS-GFP ml parasites. RHP ; to verify correct integration into the pccrmp4 locus, a 3.3kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp4. To verify deletion of pccrmp4 in wild-type and PcAS-GFP ml parasites, primers crmp4F x crmp4R were used to amplify a 700bp fragment in wild-type but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp4 but not in wild-type DNA.

    Techniques Used: Modification, Plasmid Preparation, Transfection, Amplification, Construct, Giemsa Stain

    18) Product Images from "Trends and determinants of gastric bacterial colonization of preterm neonates in a NICU setting"

    Article Title: Trends and determinants of gastric bacterial colonization of preterm neonates in a NICU setting

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114664

    Elution of DGGE bands for sequence analysis. Identification of bacterial species based on BLAST alignment (% similarity) to 16S rRNA NCBI database. Bacteria listed as “matched” showed identical DGGE band migration pattern in line with respective ATCC standards (known DNA marker). Bands not aligned with ATCC standards showed sequence homology to other Enterobacteriaceae species in the NCBI RDP database.
    Figure Legend Snippet: Elution of DGGE bands for sequence analysis. Identification of bacterial species based on BLAST alignment (% similarity) to 16S rRNA NCBI database. Bacteria listed as “matched” showed identical DGGE band migration pattern in line with respective ATCC standards (known DNA marker). Bands not aligned with ATCC standards showed sequence homology to other Enterobacteriaceae species in the NCBI RDP database.

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Sequencing, Migration, Marker

    19) Product Images from "An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts"

    Article Title: An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00583

    Exploring the role of miR-6891-5p in selective IgA deficiency (A) pedigree of affected (proband, black shadowing) and unaffected (white shadowing) family members is presented in panels B and C . (B) HLA-B , miR-6891-5p IGHA1 , and IGHA2 expression (qPCR) among IgA-deficient B-LCLs collected from affected individuals and unaffected family members (standard error bars shown, n = 3). (C) Selective IgA deficient cell line ID18 was transduced with a lentiviral construct expressing either the antisense miR-6891-5p (miR-6891-5p inhibition) or the scrambled sequence of antisense miR-6891-5p (control). Total RNA was purified, and IGHA1 and IGHA2 mRNA transcript levels were analyzed by qPCR ( y -axis shown on left of plot, standard error bars shown, n = 3). After 24 h, media was collected and analyzed by ELISA using anti-IgA antibody ( y -axis shown on right of plot, standard error bars shown, n = 3). All p -values shown are calculated using a t -test.
    Figure Legend Snippet: Exploring the role of miR-6891-5p in selective IgA deficiency (A) pedigree of affected (proband, black shadowing) and unaffected (white shadowing) family members is presented in panels B and C . (B) HLA-B , miR-6891-5p IGHA1 , and IGHA2 expression (qPCR) among IgA-deficient B-LCLs collected from affected individuals and unaffected family members (standard error bars shown, n = 3). (C) Selective IgA deficient cell line ID18 was transduced with a lentiviral construct expressing either the antisense miR-6891-5p (miR-6891-5p inhibition) or the scrambled sequence of antisense miR-6891-5p (control). Total RNA was purified, and IGHA1 and IGHA2 mRNA transcript levels were analyzed by qPCR ( y -axis shown on left of plot, standard error bars shown, n = 3). After 24 h, media was collected and analyzed by ELISA using anti-IgA antibody ( y -axis shown on right of plot, standard error bars shown, n = 3). All p -values shown are calculated using a t -test.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transduction, Construct, Inhibition, Sequencing, Purification, Enzyme-linked Immunosorbent Assay

    Validation of miR-6891-5p-mediated posttranscriptional regulation of immunoglobulin heavy chain alpha 1 and 2 ( IGHA1 and IGHA2 ) transcripts . (A) COX cells were transduced with lentiviral constructs expressing either the scrambled control or antisense sequence of miR-6891-5p. Cells were harvested after 48 h of transduction, total RNA was purified, and both IGHA1 and IGHA2 expression were analyzed by qPCR (ΔΔCt, standard error bars shown, n = 3). (B) COX cells (5 × 10 8 ) were transduced with lentiviral construct expressing either the scrambled control or antisense sequence of miR-6891-5p. After 120 h, media were collected and analyzed by ELISA using IgA antibody (standard error bars shown, n = 3). (C) Predicted binding site and heteroduplex formed between the wild-type (WT) 3′UTR of IGHA2 and miR-6891-5p. The heteroduplex formed with IGHA1 is identical to that shown. (D) Predicted binding site and heteroduplex formed between the mutated (Mut) 3′UTR sequence of IGHA2 and miR-6891-5p. (E) Either the wild-type (WT) or mutated (Mut) 3′UTR sequence of IGHA2 was cloned downstream of the luciferase reporter, creating two separate constructs. The wild-type or mutant luciferase constructs alone or together with either the miR-6891-5p expression construct (miR overexpression) or the antisense miR-6891-5p expression construct (miR inhibition) were transfected into HEK293T cells. Luciferase assay was performed 24 h after transfection (standard error bars shown, n = 3). All p -values shown are calculated using a t -test.
    Figure Legend Snippet: Validation of miR-6891-5p-mediated posttranscriptional regulation of immunoglobulin heavy chain alpha 1 and 2 ( IGHA1 and IGHA2 ) transcripts . (A) COX cells were transduced with lentiviral constructs expressing either the scrambled control or antisense sequence of miR-6891-5p. Cells were harvested after 48 h of transduction, total RNA was purified, and both IGHA1 and IGHA2 expression were analyzed by qPCR (ΔΔCt, standard error bars shown, n = 3). (B) COX cells (5 × 10 8 ) were transduced with lentiviral construct expressing either the scrambled control or antisense sequence of miR-6891-5p. After 120 h, media were collected and analyzed by ELISA using IgA antibody (standard error bars shown, n = 3). (C) Predicted binding site and heteroduplex formed between the wild-type (WT) 3′UTR of IGHA2 and miR-6891-5p. The heteroduplex formed with IGHA1 is identical to that shown. (D) Predicted binding site and heteroduplex formed between the mutated (Mut) 3′UTR sequence of IGHA2 and miR-6891-5p. (E) Either the wild-type (WT) or mutated (Mut) 3′UTR sequence of IGHA2 was cloned downstream of the luciferase reporter, creating two separate constructs. The wild-type or mutant luciferase constructs alone or together with either the miR-6891-5p expression construct (miR overexpression) or the antisense miR-6891-5p expression construct (miR inhibition) were transfected into HEK293T cells. Luciferase assay was performed 24 h after transfection (standard error bars shown, n = 3). All p -values shown are calculated using a t -test.

    Techniques Used: Transduction, Construct, Expressing, Sequencing, Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Binding Assay, Clone Assay, Luciferase, Mutagenesis, Over Expression, Inhibition, Transfection

    20) Product Images from "Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure"

    Article Title: Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure

    Journal: bioRxiv

    doi: 10.1101/2020.05.28.115972

    Cell-type specific expression with NEPTUNE and miniPromoters. (A) Transduction with hPGK-H2b-GFP (LV-GFP) at E7.5 results in widespread expression in skin as well as brain. MiniPromotor sequences for Dcx, Olig and Gfap were cloned into LV-H2b-GFP, replacing the PGK promotor, to drive expression in neuronal progenitors, oligodendrocytes or astrocytes. Transduction with Dcx-H2b-GFP resulted in obvious GFP expression in brain, with no GFP+ cells in skin. (B) Transduction with hPGK-H2b-GFP at E7.5 targets future progenitors (SOX2+), neurons (NeuN+), astrocytes (GFAP+) and oligodendrocytes (OLIG2+) at E18.5. Scale bars are 10µm. (C) MiniPromotors achieve conditional expression. None of the constructs was expressed in SOX2+ neural stem cells (white brackets). Dcx-H2b-GFP is exclusively expressed in neuronal cells, Olig1-H2b-GFP is widespread in OLIG1 positive regions, and Gfap-H2b-GFP was exclusively expressed in GFAP+ cells. Scale bars are 50µm.
    Figure Legend Snippet: Cell-type specific expression with NEPTUNE and miniPromoters. (A) Transduction with hPGK-H2b-GFP (LV-GFP) at E7.5 results in widespread expression in skin as well as brain. MiniPromotor sequences for Dcx, Olig and Gfap were cloned into LV-H2b-GFP, replacing the PGK promotor, to drive expression in neuronal progenitors, oligodendrocytes or astrocytes. Transduction with Dcx-H2b-GFP resulted in obvious GFP expression in brain, with no GFP+ cells in skin. (B) Transduction with hPGK-H2b-GFP at E7.5 targets future progenitors (SOX2+), neurons (NeuN+), astrocytes (GFAP+) and oligodendrocytes (OLIG2+) at E18.5. Scale bars are 10µm. (C) MiniPromotors achieve conditional expression. None of the constructs was expressed in SOX2+ neural stem cells (white brackets). Dcx-H2b-GFP is exclusively expressed in neuronal cells, Olig1-H2b-GFP is widespread in OLIG1 positive regions, and Gfap-H2b-GFP was exclusively expressed in GFAP+ cells. Scale bars are 50µm.

    Techniques Used: Expressing, Transduction, Clone Assay, Construct

    Knockdown of Sptbn2 in vivo with NEPTUNE reveals a novel role in neurulation and abdominal wall closure. (A) Five shRNAs against Sptbn2 were tested for knockdown efficiency in neural NE4C cells at 24 and 48 hours. (B) shRNA #1 and #2 were selected for subcloning into hPGK-H2b-GFP, and maintained knock-down efficiency was confirmed for several clones after 24 hours transfection in NE4C cells. (C) Bright field and fluorescence imaging of transfected NE4C cells confirmed GFP expression for sub-cloned shRNAs and revealed ca 50% transfection efficiency. (D) In utero transduction of embryos with shRNA#1 or shRNA#2 against Sptbn2 resulted in 87% survival at E9.5, 33% survival at E13.5 and 0% survival at E18.5. (E) At E9.5 injected embryos displayed a neurulation/turning defect with rightward skewing of the body axis (white arrowheads). (F) At E13.5, embryos continued to display halted turning, rightward skewing of the body axis (white arrowheads) and abdominal wall defects with externalized abdominal organs (black arrowheads).
    Figure Legend Snippet: Knockdown of Sptbn2 in vivo with NEPTUNE reveals a novel role in neurulation and abdominal wall closure. (A) Five shRNAs against Sptbn2 were tested for knockdown efficiency in neural NE4C cells at 24 and 48 hours. (B) shRNA #1 and #2 were selected for subcloning into hPGK-H2b-GFP, and maintained knock-down efficiency was confirmed for several clones after 24 hours transfection in NE4C cells. (C) Bright field and fluorescence imaging of transfected NE4C cells confirmed GFP expression for sub-cloned shRNAs and revealed ca 50% transfection efficiency. (D) In utero transduction of embryos with shRNA#1 or shRNA#2 against Sptbn2 resulted in 87% survival at E9.5, 33% survival at E13.5 and 0% survival at E18.5. (E) At E9.5 injected embryos displayed a neurulation/turning defect with rightward skewing of the body axis (white arrowheads). (F) At E13.5, embryos continued to display halted turning, rightward skewing of the body axis (white arrowheads) and abdominal wall defects with externalized abdominal organs (black arrowheads).

    Techniques Used: In Vivo, shRNA, Subcloning, Clone Assay, Transfection, Fluorescence, Imaging, Expressing, In Utero, Transduction, Injection

    21) Product Images from "A novel ribosomal protein S20 variant in a family with unexplained colorectal cancer and polyposis"

    Article Title: A novel ribosomal protein S20 variant in a family with unexplained colorectal cancer and polyposis

    Journal: bioRxiv

    doi: 10.1101/2019.12.16.877084

    Confirmation of splicing defect. A) RPS20 NM_001023.3 transcript with variants linked to CRC. c.147dupA (Nieminen et al, 2014). c.177+1G > A (this study). c.181_182delTT (Broderick et al, 2017). B) RT-PCR of RPS20 from Lynch syndrome, FAP, and RPS20 c.177+1G > A subjects. FAP (familial adenomatous polyposis); NTC (no template control); NRTC (no reverse transcriptase control). C) 591bp band corresponding to partial intron 3 inclusion. Black arrow indicates c.177+1G > A variant position. Note the chromatogram shows only adenine. D) 444bp band corresponding to wild-type NM_001023.3 sequence. E) 370bp band corresponding to exon 3 skipping.
    Figure Legend Snippet: Confirmation of splicing defect. A) RPS20 NM_001023.3 transcript with variants linked to CRC. c.147dupA (Nieminen et al, 2014). c.177+1G > A (this study). c.181_182delTT (Broderick et al, 2017). B) RT-PCR of RPS20 from Lynch syndrome, FAP, and RPS20 c.177+1G > A subjects. FAP (familial adenomatous polyposis); NTC (no template control); NRTC (no reverse transcriptase control). C) 591bp band corresponding to partial intron 3 inclusion. Black arrow indicates c.177+1G > A variant position. Note the chromatogram shows only adenine. D) 444bp band corresponding to wild-type NM_001023.3 sequence. E) 370bp band corresponding to exon 3 skipping.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Sequencing

    22) Product Images from "Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing"

    Article Title: Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1711158114

    RSF1 binds H2Aub nucleosomes through a previously uncharacterized UAB domain. ( A ) Schematic representation of the RSF1 domain organization and fragments used in nucleosome pull-down assay. ( B ) The full-length and fragment 7 (amino acids 770–807) of RSF1 bind to H2Aub nucleosomes. ( Top ) CBB-stained protein gel containing purified full-length RSF1 and its serial fragments. F1: 1–97 aa; F2, 98–182 aa; F3, 183–397 aa; F4, 398–608 aa; F5, 609–690 aa; F6, 691–785 aa; F7, 770–807 aa; F8, 808–890 aa; F9, 891–941 aa; F10, 942–1,441 aa. ( Middle ) Pull-down assays show that only the full-length RSF1 and fragment 7 (designated as the UAB domain) bind to H2Aub nucleosomes. ( Bottom ) Treatment with the H2Aub-specific deubiquitinase USP16 abolishes binding of RSF1 and fragment 7 to the nucleosomes. ( C ) The UAB domain specifically pulls down reconstituted nucleosomes subjected to in vitro ubiquitination by PRC1 (lane 2), but not unmodified nucleosomes (lane 1). ( D ) The UAB domain specifically pulls down H2Aub (lane 1), but not H2Bub (lane 2) nucleosomes. ( E ) The UAB domain is required for RSF1 to interact with H2Aub nucleosomes in 293T cells. Cells were transfected with an empty vector or vectors expressing Flag-RSF1 or mutated Flag-RSF1 (MT) in which the UAB domain was in-frame deleted. Mononucleosomes were prepared and subjected to anti-Flag IP followed by immunoblots. ( F ) Metaplot of Flag-RSF1 in control (blue line) and Ring1B KO (red line) mouse ESCs. For all data, the minimum value is set to 0. The signal plot is normalized by subtracting IgG ChIP-seq signal from the same cell lines. Transcription start site (TSS) plus 10-kb upstream and transcription termination site (TTS) plus 10-kb downstream are shown. ( G ) Venn diagram shows the overlap between RSF1 bound genes and H2Aub marked genes in mouse ESCs. About 82% of H2Aub sites are bound by RSF1.
    Figure Legend Snippet: RSF1 binds H2Aub nucleosomes through a previously uncharacterized UAB domain. ( A ) Schematic representation of the RSF1 domain organization and fragments used in nucleosome pull-down assay. ( B ) The full-length and fragment 7 (amino acids 770–807) of RSF1 bind to H2Aub nucleosomes. ( Top ) CBB-stained protein gel containing purified full-length RSF1 and its serial fragments. F1: 1–97 aa; F2, 98–182 aa; F3, 183–397 aa; F4, 398–608 aa; F5, 609–690 aa; F6, 691–785 aa; F7, 770–807 aa; F8, 808–890 aa; F9, 891–941 aa; F10, 942–1,441 aa. ( Middle ) Pull-down assays show that only the full-length RSF1 and fragment 7 (designated as the UAB domain) bind to H2Aub nucleosomes. ( Bottom ) Treatment with the H2Aub-specific deubiquitinase USP16 abolishes binding of RSF1 and fragment 7 to the nucleosomes. ( C ) The UAB domain specifically pulls down reconstituted nucleosomes subjected to in vitro ubiquitination by PRC1 (lane 2), but not unmodified nucleosomes (lane 1). ( D ) The UAB domain specifically pulls down H2Aub (lane 1), but not H2Bub (lane 2) nucleosomes. ( E ) The UAB domain is required for RSF1 to interact with H2Aub nucleosomes in 293T cells. Cells were transfected with an empty vector or vectors expressing Flag-RSF1 or mutated Flag-RSF1 (MT) in which the UAB domain was in-frame deleted. Mononucleosomes were prepared and subjected to anti-Flag IP followed by immunoblots. ( F ) Metaplot of Flag-RSF1 in control (blue line) and Ring1B KO (red line) mouse ESCs. For all data, the minimum value is set to 0. The signal plot is normalized by subtracting IgG ChIP-seq signal from the same cell lines. Transcription start site (TSS) plus 10-kb upstream and transcription termination site (TTS) plus 10-kb downstream are shown. ( G ) Venn diagram shows the overlap between RSF1 bound genes and H2Aub marked genes in mouse ESCs. About 82% of H2Aub sites are bound by RSF1.

    Techniques Used: Pull Down Assay, Staining, Purification, Binding Assay, In Vitro, Transfection, Plasmid Preparation, Expressing, Western Blot, Chromatin Immunoprecipitation

    23) Product Images from "Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine"

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine

    Journal: Nature protocols

    doi: 10.1038/nprot.2012.137

    Overview of Tet-assisted bisulfite sequencing (TAB-seq). 5-hmC is protected specifically by β-GT to generate 5-gmC, followed by oxidation of 5-mC to 5-caC by mTet1. Only 5-gmC is read as C after bisulfite treatment and PCR amplification.
    Figure Legend Snippet: Overview of Tet-assisted bisulfite sequencing (TAB-seq). 5-hmC is protected specifically by β-GT to generate 5-gmC, followed by oxidation of 5-mC to 5-caC by mTet1. Only 5-gmC is read as C after bisulfite treatment and PCR amplification.

    Techniques Used: Methylation Sequencing, Polymerase Chain Reaction, Amplification

    24) Product Images from "A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain"

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    Journal: eLife

    doi: 10.7554/eLife.43322

    Cloning of V L and V H domain sequences from hybridomas into the R-mAb expression plasmid. ( A ) Agarose gel analysis of V L and V H domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.
    Figure Legend Snippet: Cloning of V L and V H domain sequences from hybridomas into the R-mAb expression plasmid. ( A ) Agarose gel analysis of V L and V H domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Synthesized, Derivative Assay

    Cloning of anti-Kv2.1 D3/71 V L and V H domain cDNAs from a nonviable hybridoma. ( A ) Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈360 bp, and of the cleaved aberrant V L domain is ≈180 bp. ( B ) Agarose gel analysis of D3/71 V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316 joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp.
    Figure Legend Snippet: Cloning of anti-Kv2.1 D3/71 V L and V H domain cDNAs from a nonviable hybridoma. ( A ) Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈360 bp, and of the cleaved aberrant V L domain is ≈180 bp. ( B ) Agarose gel analysis of D3/71 V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316 joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp.

    Techniques Used: Clone Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

    25) Product Images from "Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation"

    Article Title: Dynamic Methylation of an L1 Transduction Family during Reprogramming and Neurodifferentiation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00499-18

    Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.
    Figure Legend Snippet: Characterization of a reprogramming-associated de novo L1 insertion carried through neurodifferentiation in vitro . (A) Schematic timeline of experimental approach. Fibroblasts (time point 0 [ T 0 ]) were reprogrammed to obtain hiPSCs ( T 1 ), which were then sampled at 5 points ( T 2 to T 6 ) of neuronal differentiation in extended cell culture. Immunocytochemistry was used to characterize expression of marker genes (OCT4, NANOG, PAX6, TUJ1, CUX1, and GFAP gemes) and histone 3 phosphorylation (PH3), as associated with various stages of neural cell maturation, with Hoechst staining of DNA. (B) L1 insertion PCR validation strategies. Green and blue arrows, respectively, represent primers targeting the 5′ and 3′ genomic flanks of an L1 insertion (rectangle). Black arrows represent primers specific to the L1 sequence. Combinations of these primers are used to generate the following amplicons (arranged top to bottom): 5′ L1-genome junction, 3′ L1-genome junction, L1 insertion (filled site), and empty site. (C) PCR validation results for a de novo L1 insertion detected in cell line hiPSC-CRL2429. An empty/filled PCR was also performed with cell line hiPSC-CRL1502 as a negative control. Red and black arrow heads indicate the expected filled and empty site band sizes, respectively. NTC, nontemplate control. (D) De novo L1 insertion sequence structure. In addition to TSDs (triangles), the full-length L1-Ta insertion was flanked by 5′ (orange) and 3′ transductions (purple). (E) The same experiments as described for panel C except that they were performed for the donor L1 responsible for the de novo L1 insertion (left) and its lineage progenitor L1 (right), using CRL2429 fibroblast genomic DNA.

    Techniques Used: In Vitro, Cell Culture, Immunocytochemistry, Expressing, Marker, Staining, Polymerase Chain Reaction, Sequencing, Negative Control

    L1 promoter DNA methylation is dynamic during hiPSC-CRL2429 reprogramming and neurodifferentiation. (A) L1 bisulfite sequencing analysis design. CpG dinucleotides are indicated by circles above the L1 5ʹ UTR, and their nucleotide positions are provided below. A common reverse primer (black) is combined with either an L1-Ta subfamily forward primer (purple) or an L1 locus-specific forward primer (pink) to generate PCR amplicons for multiplexed paired-end Illumina 2- by 300-mer sequencing, resolving each amplicon in full. (B) L1 CpG methylation patterns in hiPSC-CRL2429 fibroblasts, hiPSCs, and neural cells derived in vitro . Each cartoon panel corresponds to an amplicon (L1-Ta subfamily or specific L1 locus) and displays 50 random, nonidentical sequences (black circle, methylated CpG; white circle, unmethylated CpG; ×, mutated CpG). The percentage of methylated CpG is indicated in the lower right corner of each cartoon. (C) L1 promoter CpG methylation levels for the hiPSC-CRL2429 neurodifferentiation time course. Values represent the means ± standard deviations of CpG methylation of the corresponding 50 reads for each amplicon, as presented in panel B. Statistical analyses involved paired t tests, with a Bonferroni multiple-testing correction where appropriate. *, P
    Figure Legend Snippet: L1 promoter DNA methylation is dynamic during hiPSC-CRL2429 reprogramming and neurodifferentiation. (A) L1 bisulfite sequencing analysis design. CpG dinucleotides are indicated by circles above the L1 5ʹ UTR, and their nucleotide positions are provided below. A common reverse primer (black) is combined with either an L1-Ta subfamily forward primer (purple) or an L1 locus-specific forward primer (pink) to generate PCR amplicons for multiplexed paired-end Illumina 2- by 300-mer sequencing, resolving each amplicon in full. (B) L1 CpG methylation patterns in hiPSC-CRL2429 fibroblasts, hiPSCs, and neural cells derived in vitro . Each cartoon panel corresponds to an amplicon (L1-Ta subfamily or specific L1 locus) and displays 50 random, nonidentical sequences (black circle, methylated CpG; white circle, unmethylated CpG; ×, mutated CpG). The percentage of methylated CpG is indicated in the lower right corner of each cartoon. (C) L1 promoter CpG methylation levels for the hiPSC-CRL2429 neurodifferentiation time course. Values represent the means ± standard deviations of CpG methylation of the corresponding 50 reads for each amplicon, as presented in panel B. Statistical analyses involved paired t tests, with a Bonferroni multiple-testing correction where appropriate. *, P

    Techniques Used: DNA Methylation Assay, Methylation Sequencing, Polymerase Chain Reaction, Sequencing, Amplification, CpG Methylation Assay, Derivative Assay, In Vitro, Methylation

    26) Product Images from "Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1"

    Article Title: Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

    Journal: Scientific Reports

    doi: 10.1038/srep25802

    SLC20A2 Knock Down ablates calcitriol-mediated blockade of calcification. SaOs-2 cells were transfected with guide-RNA against SLC20A2 together with Cas-9. ( A ) Gene knockdown was confirmed by PCR amplification of the targeted region and T7 digests to confirm site cutting. Note the reduction in ~500 bp fragments and the appearance of shorter fragments in the CRISPR cells, which is the result of mismatch pairing of the DNA after repair by non-homologous end joining. ( B ) Primers were designed to flank the guide-RNA targeting site and genomic PCR performed on DNA from cells transfected with a donor plasmid (containing a Puromycin resistance gene flanked by homologous arms complimentary to the gene insertion site) with Cas-9. The reduced presence of a ~500 bp fragment is caused by the puromycin insertion, extending the size of the amplicon, thereby producing a diminished band in the PCR reaction. ( C ) Western blot analysis confirmed that protein expression was reduced in CRISPR-treated cells (representative of n = 2). ( D ) Wild type (WT) or SLC20A2 knock down (KD) SaOs-2 cells were treated for 14 days under calcifying conditions with or without calcitriol (Calc). KD cells were no longer protected from calcification by calcitriol (n = 3 independent experiments, *p
    Figure Legend Snippet: SLC20A2 Knock Down ablates calcitriol-mediated blockade of calcification. SaOs-2 cells were transfected with guide-RNA against SLC20A2 together with Cas-9. ( A ) Gene knockdown was confirmed by PCR amplification of the targeted region and T7 digests to confirm site cutting. Note the reduction in ~500 bp fragments and the appearance of shorter fragments in the CRISPR cells, which is the result of mismatch pairing of the DNA after repair by non-homologous end joining. ( B ) Primers were designed to flank the guide-RNA targeting site and genomic PCR performed on DNA from cells transfected with a donor plasmid (containing a Puromycin resistance gene flanked by homologous arms complimentary to the gene insertion site) with Cas-9. The reduced presence of a ~500 bp fragment is caused by the puromycin insertion, extending the size of the amplicon, thereby producing a diminished band in the PCR reaction. ( C ) Western blot analysis confirmed that protein expression was reduced in CRISPR-treated cells (representative of n = 2). ( D ) Wild type (WT) or SLC20A2 knock down (KD) SaOs-2 cells were treated for 14 days under calcifying conditions with or without calcitriol (Calc). KD cells were no longer protected from calcification by calcitriol (n = 3 independent experiments, *p

    Techniques Used: Transfection, Polymerase Chain Reaction, Amplification, CRISPR, Non-Homologous End Joining, Plasmid Preparation, Western Blot, Expressing

    27) Product Images from "Human GM3 Synthase Attenuates Taxol-Triggered Apoptosis Associated with Downregulation of Caspase-3 in Ovarian Cancer Cells"

    Article Title: Human GM3 Synthase Attenuates Taxol-Triggered Apoptosis Associated with Downregulation of Caspase-3 in Ovarian Cancer Cells

    Journal: Journal of cancer therapy

    doi: 10.4236/jct.2012.35065

    GM3 synthase prevents caspase-3 activation. Western blot analysis of GM3 synthase expression in SKOV3/GS cells using V5 antibody. SKOV3 and SKOV3/GS cells were treated with 100 nM Taxol for 48 h and GM3 synthase was detected by Western blot analysis using
    Figure Legend Snippet: GM3 synthase prevents caspase-3 activation. Western blot analysis of GM3 synthase expression in SKOV3/GS cells using V5 antibody. SKOV3 and SKOV3/GS cells were treated with 100 nM Taxol for 48 h and GM3 synthase was detected by Western blot analysis using

    Techniques Used: Activation Assay, Western Blot, Expressing

    Taxol-induced apoptosis and GM3 synthase expression in SKOV3 cells. (a) Cells were treated with 100 nM Taxol for 48 h, harvested, and apoptosis was determined by FACS analysis as described in Materials and Methods. Compensation was executed for each experiment
    Figure Legend Snippet: Taxol-induced apoptosis and GM3 synthase expression in SKOV3 cells. (a) Cells were treated with 100 nM Taxol for 48 h, harvested, and apoptosis was determined by FACS analysis as described in Materials and Methods. Compensation was executed for each experiment

    Techniques Used: Expressing, FACS

    GM3 synthase increases proliferation and decreases Taxol cytotoxicity in SKOV3/GS cells. (a) SKOV3 and SKOV3/GS cells were grown for 48 h and the number of cells was determined as described in Materials and Methods. (b) SKOV3 and SKOV3/GS cells were treated
    Figure Legend Snippet: GM3 synthase increases proliferation and decreases Taxol cytotoxicity in SKOV3/GS cells. (a) SKOV3 and SKOV3/GS cells were grown for 48 h and the number of cells was determined as described in Materials and Methods. (b) SKOV3 and SKOV3/GS cells were treated

    Techniques Used:

    28) Product Images from "Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation"

    Article Title: Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbagrm.2018.03.007

    Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p
    Figure Legend Snippet: Effect of SAHA on histone modifications associated with the COX - 2 promoter. A, Schematic representation of COX - 2 promoter region identifying single CpG sites (vertical bar), binding sites for transcription factors NF-κB (Nuclear factor-κB), C/EBP (CCAAT/enhancer binding protein) and CRE (cAMP-response element), transcription start site (+1), translational coding site ATG and regions amplified by ChIP primers (ChIP-set A and ChIP-set B). B–E, F-NL from 3 donors were pre-treated with SAHA (5 μM) for 1 h prior to incubation with TGF-β1 (2 ng/ml) for 72 h. ChIP assay was performed using antibodies against acetylated histone H3 (B, C) and H3K27me3 (D, E) and the associated COX - 2 promoter DNA was detected by real-time PCR using ChIP-set A (B, D) and ChIP-set B (C, E) primers. Data are normalized to the input control and reported as mean ± SEM of three biological replicates. *p

    Techniques Used: Binding Assay, Amplification, Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction

    29) Product Images from "Fluorescence-based methods for measuring target interference by CRISPR-Cas systems"

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2018.10.027

    Development of pACYC-GFP. A. Constitutive promoter sequences tested during construction of pACYC-GFP. Promoters 1–4 are variants of tac promoter that reduce the promoter strength. Promoter 1 contains an extra base pair between the −10 and −35 sites. Promoters 2, 3 and 4 contain one, two or three variations in the −35 site, respectively. Variations between promoters are underlined. Gaps in aligned promoter sequences are represented with spaces. B. Flow cytometry histograms for the five promoters tested. p1-p4: promoters 1–4. C. Competition assays between cells harboring pACYCDuet-1 (GFP−) and pACYC-GFP (GFP+). The population distribution remains the same after 24 h of growth, indicating that pACYC-GFP does not affect growth rate of the cells.
    Figure Legend Snippet: Development of pACYC-GFP. A. Constitutive promoter sequences tested during construction of pACYC-GFP. Promoters 1–4 are variants of tac promoter that reduce the promoter strength. Promoter 1 contains an extra base pair between the −10 and −35 sites. Promoters 2, 3 and 4 contain one, two or three variations in the −35 site, respectively. Variations between promoters are underlined. Gaps in aligned promoter sequences are represented with spaces. B. Flow cytometry histograms for the five promoters tested. p1-p4: promoters 1–4. C. Competition assays between cells harboring pACYCDuet-1 (GFP−) and pACYC-GFP (GFP+). The population distribution remains the same after 24 h of growth, indicating that pACYC-GFP does not affect growth rate of the cells.

    Techniques Used: Flow Cytometry, Cytometry

    Measurement of Cas9 cleavage using GFP reporter assay. A. Schematic of cas9 and gRNA expression plasmids. The cas9 gene and sgRNA are both expressed from arabinose inducible pBAD promoters. B. Target inserted into pACYC-GFP for this study. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. EcoRI and NotI overhangs are labeled. Positions of G3A or G7T (non-target strand sequence) mutations are indicated. C. Plasmid loss assay for Cas9 targets containing a perfect sequence or seed mismatches at the third or seventh position. Empty pACYC-GFP (no target) was used as a control to ensure that the plasmid is stable in the absence of CRISPR interference. Plasmid loss (%) is the percentage of GFP- cells based on flow cytometry measurements at each time point. The average plasmid loss from 3 replicates is shown, with error representing standard deviation.
    Figure Legend Snippet: Measurement of Cas9 cleavage using GFP reporter assay. A. Schematic of cas9 and gRNA expression plasmids. The cas9 gene and sgRNA are both expressed from arabinose inducible pBAD promoters. B. Target inserted into pACYC-GFP for this study. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. EcoRI and NotI overhangs are labeled. Positions of G3A or G7T (non-target strand sequence) mutations are indicated. C. Plasmid loss assay for Cas9 targets containing a perfect sequence or seed mismatches at the third or seventh position. Empty pACYC-GFP (no target) was used as a control to ensure that the plasmid is stable in the absence of CRISPR interference. Plasmid loss (%) is the percentage of GFP- cells based on flow cytometry measurements at each time point. The average plasmid loss from 3 replicates is shown, with error representing standard deviation.

    Techniques Used: Reporter Assay, Expressing, Labeling, Sequencing, Plasmid Preparation, CRISPR, Flow Cytometry, Cytometry, Standard Deviation

    Design of fluorescence-based CRISPR interference assay. A. Schematic of Cas-crRNA effector complex bound to dsDNA. The spacer-protospacer RNA:DNA hybrid is shown in yellow. The PAM is highlighted in red. The PAM-proximal seed region is labeled. B. Schematic of pACYC-GFP plasmid. MCS1: multiple cloning site 1. C. Close-up schematic of GFP expression cassette in pACYC-GFP. The locations of the constitutive promoter and ssrA degradation tag are highlighted. D. Basis for fluorescence-based plasmid loss assay. As the plasmid concentration decreases due to CRISPR-based plasmid loss, the cells become less fluorescent.
    Figure Legend Snippet: Design of fluorescence-based CRISPR interference assay. A. Schematic of Cas-crRNA effector complex bound to dsDNA. The spacer-protospacer RNA:DNA hybrid is shown in yellow. The PAM is highlighted in red. The PAM-proximal seed region is labeled. B. Schematic of pACYC-GFP plasmid. MCS1: multiple cloning site 1. C. Close-up schematic of GFP expression cassette in pACYC-GFP. The locations of the constitutive promoter and ssrA degradation tag are highlighted. D. Basis for fluorescence-based plasmid loss assay. As the plasmid concentration decreases due to CRISPR-based plasmid loss, the cells become less fluorescent.

    Techniques Used: Fluorescence, CRISPR, Labeling, Plasmid Preparation, Clone Assay, Expressing, Concentration Assay

    Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.
    Figure Legend Snippet: Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Techniques Used: CRISPR, Sequencing, Mutagenesis, Labeling

    30) Product Images from "Tuning of mRNA stability through altering 3′-UTR sequences generates distinct output expression in a synthetic circuit driven by p53 oscillations"

    Article Title: Tuning of mRNA stability through altering 3′-UTR sequences generates distinct output expression in a synthetic circuit driven by p53 oscillations

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-42509-y

    Distinct naturally occurring 3′-UTRs confer different output transcript stabilities to generate different expression dynamics of mCherry expression induced by p53 oscillations. ( A ) Schematic of the synthetic circuit with output reporter constructs with DDB2 , TRIAP1 or GADD45A 3′-UTR inserted following the mCherry coding sequence. Predicted phenotype for output mCherry expression in individual cells. ( B – D ) mRNA decay rates of mCherry transcripts with the DDB2 ( B ), TRIAP1 ( C ), or GADD45A ( D ) 3′-UTR. All data are normalized by GAPDH transcript levels. Data are mean values ± SEM for 5 biological replicates. ( E – J ) mCherry expression in single cells following NCS treatment to induce p53 oscillations in cells containing DDB2 ( E , F ), TRIAP1 ( G , H ), or GADD45A ( I , J ) 3′-UTR for the mCherry output transcript. Gray lines represent mean nuclear mCherry expression in individual cells, red lines represent mean of the population for all cells in each condition ( E , G , I ). Representative single cell traces are shown in ( F , H , J ). Approximately 50 cells were quantified per condition.
    Figure Legend Snippet: Distinct naturally occurring 3′-UTRs confer different output transcript stabilities to generate different expression dynamics of mCherry expression induced by p53 oscillations. ( A ) Schematic of the synthetic circuit with output reporter constructs with DDB2 , TRIAP1 or GADD45A 3′-UTR inserted following the mCherry coding sequence. Predicted phenotype for output mCherry expression in individual cells. ( B – D ) mRNA decay rates of mCherry transcripts with the DDB2 ( B ), TRIAP1 ( C ), or GADD45A ( D ) 3′-UTR. All data are normalized by GAPDH transcript levels. Data are mean values ± SEM for 5 biological replicates. ( E – J ) mCherry expression in single cells following NCS treatment to induce p53 oscillations in cells containing DDB2 ( E , F ), TRIAP1 ( G , H ), or GADD45A ( I , J ) 3′-UTR for the mCherry output transcript. Gray lines represent mean nuclear mCherry expression in individual cells, red lines represent mean of the population for all cells in each condition ( E , G , I ). Representative single cell traces are shown in ( F , H , J ). Approximately 50 cells were quantified per condition.

    Techniques Used: Expressing, Construct, Sequencing

    Restoration of apoptotic activity in a tunable synthetic circuit driven by p53 oscillations. ( A ) Schematic of the synthetic circuit with caspase-3 output constructs with DDB2 , TRIAP1 or GADD45A 3′-UTR inserted following the CASP3 coding sequence. Predicted phenotype for apoptosis in individual cells. (B-D) mRNA decay rates of CASP3 transcripts with the DDB2 ( B ), TRIAP1 ( C ), or GADD45A ( D ) 3′-UTR. All data are normalized by GAPDH transcript levels. Data are mean values ± SEM for 3 ( B ) or 5 ( C , D ) biological replicates. ( E ) Fold changes in the number of late apoptotic cells 24 h after NCS treatment for cells expression CASP3 with different 3′-UTRs. Data represent mean ± SEM for triplicate experiments. (*indicates statistically significant difference, p
    Figure Legend Snippet: Restoration of apoptotic activity in a tunable synthetic circuit driven by p53 oscillations. ( A ) Schematic of the synthetic circuit with caspase-3 output constructs with DDB2 , TRIAP1 or GADD45A 3′-UTR inserted following the CASP3 coding sequence. Predicted phenotype for apoptosis in individual cells. (B-D) mRNA decay rates of CASP3 transcripts with the DDB2 ( B ), TRIAP1 ( C ), or GADD45A ( D ) 3′-UTR. All data are normalized by GAPDH transcript levels. Data are mean values ± SEM for 3 ( B ) or 5 ( C , D ) biological replicates. ( E ) Fold changes in the number of late apoptotic cells 24 h after NCS treatment for cells expression CASP3 with different 3′-UTRs. Data represent mean ± SEM for triplicate experiments. (*indicates statistically significant difference, p

    Techniques Used: Activity Assay, Construct, Sequencing, Expressing

    An oscillating transcription factor generates distinct output expression dynamics as a function of mRNA decay rates. A model predicts that distinct mRNA and protein expression dynamics can be generated by altering the transcript stability of target genes induced by an oscillating transcription factor. 3′-UTRs can confer different transcript stabilities. Based on the relationship between the transcript decay rate and the oscillator frequency, output gene transcripts will have rising, weakly pulsing, or strongly pulsing mRNA expression dynamics, leading to alterations in the rate of accumulation of an output protein product.
    Figure Legend Snippet: An oscillating transcription factor generates distinct output expression dynamics as a function of mRNA decay rates. A model predicts that distinct mRNA and protein expression dynamics can be generated by altering the transcript stability of target genes induced by an oscillating transcription factor. 3′-UTRs can confer different transcript stabilities. Based on the relationship between the transcript decay rate and the oscillator frequency, output gene transcripts will have rising, weakly pulsing, or strongly pulsing mRNA expression dynamics, leading to alterations in the rate of accumulation of an output protein product.

    Techniques Used: Expressing, Generated

    31) Product Images from "Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis"

    Article Title: Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027756

    Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.
    Figure Legend Snippet: Amino acid sequence alignment of the 8 different αF1 scFv. 36% of the 252 clones that were screened by ELISA specifically bound recombinant F1 antigen. 62 of the 90 positive clones were successfully sequenced. Eight different αF1 scFv groups were identified following alignment of the full-length amino acid sequences obtained by translating the DNA sequences. One clone per group was selected. Here presented are the amino acid sequences of the selected clones. Letters indicated in the following combination of foreground/background: black/white, blue/torquoise, black/green, red/yellow and green/white, correspond to amino acids that are non-similar, conservative, similar, identical or weakly similar respectively. The consensus sequence corresponds to amino acids that are represented at least in 3 of the 8 sequences. The boxed portions of the consensus correspond to the CDRs (Kabat definition). With the red, green, blue, orange, black and pink boxes defining CDRL1 through 3 and CDRH 1 through 3 respectively.

    Techniques Used: Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant

    Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Binding analysis of αF1 scFv to recombinant F1 antigen. A ) ELISA analysis: The αF1 scFv proteins were expressed as alkaline phosphatase (AP) fusion proteins, recovered from the periplasmic fraction, and tested by one-step ELISA for binding to either recombinant F1 or chicken Lysozyme, without further purification. The binding profile of each αF1 scFv clone presented, was not normalized for expression. B ) Bead-based flow cytometry analysis: The αF1 scFv proteins were expressed as AP-Ecoil fusion proteins, recovered from the periplasmic fraction and directly labeled with Kcoil-A488. The fluorescently labeled αF1 were tested for binding to recombinant biotinylated F1 and ubiquitin by multiplex bead-based flow cytometry, without further purification. The binding profile of each αF1-APEcoil scFv clone presented was not normalized for expression. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Expressing, Flow Cytometry, Cytometry, Labeling, Multiplex Assay, Standard Deviation

    Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Cell-based flow cytometry analysis: Fluorescent aF1 phage reactivity with fixed Yersinia cells. A ) Reactivity of all aF1 scFv phage clones with 3 fixed Yersinia strains: F1-positive Y. pestis A1122 and F1-negative strains Y. enterocolytica 0107 and Y. pseudotubeculosis 0104 were incubated with either αLysozyme (CTD1.3) or αF1 scFv-displaying FITC-labeled phage (CT1 through 8). The fluorescence associated with each cell type was measured using FACS Calibur and data were analyzed by CellQuest. B ) Reactivity of αF1 CT8 and CTD1.3 with 8 fixed Yersinia strains. F1-positive Y. pestis strains A1122, C092, India, India 15 and Kim, and F1-negative strains Y. pestis Nairobi, Y. enterocolytica 0107 and Y. pseudotuberculosis 0104 were incubated with FITC-labeled phage. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Clone Assay, Incubation, Labeling, Fluorescence, FACS, Standard Deviation

    Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Whole-cell ELISA analysis: aF1 phage reactivity with live or fixed Yersinia cells . Each phage-displayed αF1 (CT1 through 8) or αLysozyme (CTD1.3) scFv was incubated with blocked live ( A ) or fixed ( B ) F1-positive Yersinia pestis (YP) A1122, Kim, India and India 195 or F1-negative YP Nairobi, Yersinia pseudotuberculosis 0104 (YPT 0104) and Yersinia enterocolytica 0107 (YE 0107). Phage-binding events were reported using αM13-HRP antibody. Background noise coming from buffers, secondary antibody or the cells was evaluated by including wells with no added phage (no phage). The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay, Standard Deviation

    aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.
    Figure Legend Snippet: aF1 CT phage are stable, and reactive, following prolonged storage. Phage-displayed αF1 scFv CT4, CT6 and CT5 (inactive control) were treated with a preservative solution and tested for reactivity following different storage conditions; A) freshly prepared phage, B) 6 months at 4°C, C) 9 months at 4°C followed by 1 month at room temperature (RT) and D) 9 months at 4°C followed by 2 months at RT. Phage was tested for activity at non-saturating concentrations by whole-cell ELISA using live Yersinia pestis A1122 cells. Each value is an average of 3 experiments with corresponding standard deviation.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.
    Figure Legend Snippet: Bead-based flow cytometry analysis: aF1 phage reactivity with recombinant F1 antigen. A ) Schematic of analysis: a set of 3 distinct luminex beads was bound to biotinylated Lysozyme, biotinylated F1 or biotin respectively. Bound phage was stained with αM13 mouse IgG and phycoerythrin (PE)-conjugated goat αMouse. Beads were separated based on their intrinsic fluorescence (APC-A, APC-cyt7), and the associated PE stain was measured to assess specificity of binding to F1 antigen. B ) Assay results: Eight different αF1 scFv were expressed in phage format (CT1 through 8). Phage preparations were normalized to a concentration of 5×10 +12 cfu/mL and analyzed for specific binding. The value associated to each bar is an average of three experiments with corresponding standard deviation.

    Techniques Used: Flow Cytometry, Cytometry, Recombinant, Luminex, Staining, Fluorescence, Binding Assay, Concentration Assay, Standard Deviation

    32) Product Images from "Divergent lncRNA GATA3-AS1 Regulates GATA3 Transcription in T-Helper 2 Cells"

    Article Title: Divergent lncRNA GATA3-AS1 Regulates GATA3 Transcription in T-Helper 2 Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02512

    GATA3-AS1 is localized in the nucleus and expression increases during TH2 cell polarization . (A) Cellular and nuclear fractions of primary and effector TH2 populations analyzed via qPCR. Values represent ΔΔCT vs. whole cell GAPDH. VIM-AS1 lncRNA is a nuclear control, while HPRT mRNA represents a cytoplasm specific control. Statistical significance vs. relative cytoplasmic fraction was determined by Students T-test ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 is localized in the nucleus and expression increases during TH2 cell polarization . (A) Cellular and nuclear fractions of primary and effector TH2 populations analyzed via qPCR. Values represent ΔΔCT vs. whole cell GAPDH. VIM-AS1 lncRNA is a nuclear control, while HPRT mRNA represents a cytoplasm specific control. Statistical significance vs. relative cytoplasmic fraction was determined by Students T-test ( n = 3). * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    GATA3-AS1 RNA forms an R-Loop with the central intron of GATA3-AS1 . (A) Predictive R-Loop formations according to the R-Loop database. The largest and most likely R-Loop is circled, located between regions 2 and 3 of GATA3-AS1 . (B) DRIP assay completed with RNA isolation. Results are expressed as fold enrichment relative to the RNase H negative control. Chromatin was treated with RNase to remove R-Loops as an additional control, mean ± S.D. ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 RNA forms an R-Loop with the central intron of GATA3-AS1 . (A) Predictive R-Loop formations according to the R-Loop database. The largest and most likely R-Loop is circled, located between regions 2 and 3 of GATA3-AS1 . (B) DRIP assay completed with RNA isolation. Results are expressed as fold enrichment relative to the RNase H negative control. Chromatin was treated with RNase to remove R-Loops as an additional control, mean ± S.D. ( n = 3). * P

    Techniques Used: Isolation, Negative Control

    GATA3-AS1 is expressed in TH2 cells. (A) Genomic locations of human GATA3-AS1 known isoforms and GATA3 with directions of transcription in sense ( > ) and antisense orientations (
    Figure Legend Snippet: GATA3-AS1 is expressed in TH2 cells. (A) Genomic locations of human GATA3-AS1 known isoforms and GATA3 with directions of transcription in sense ( > ) and antisense orientations (

    Techniques Used:

    GATA3-AS1 and GATA3 form a necessary feed forward loop for TH2 polarization. (A) PBMCs were cultured under TH2 polarizing conditions for 2 days and transfected with a GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). GATA3-AS1, GATA3, IL4, IL5 , and IL13 transcripts were determined by qPCR on day 5 and results expressed relative to GAPDH . Statistical significance was determined using Students T -test by comparing GATA3-AS1 siRNA knockdown to scrambled control knockdown ( n = 3). * P
    Figure Legend Snippet: GATA3-AS1 and GATA3 form a necessary feed forward loop for TH2 polarization. (A) PBMCs were cultured under TH2 polarizing conditions for 2 days and transfected with a GATA3-AS1 specific siRNA (+) or scrambled siRNA (–). GATA3-AS1, GATA3, IL4, IL5 , and IL13 transcripts were determined by qPCR on day 5 and results expressed relative to GAPDH . Statistical significance was determined using Students T -test by comparing GATA3-AS1 siRNA knockdown to scrambled control knockdown ( n = 3). * P

    Techniques Used: Cell Culture, Transfection, Real-time Polymerase Chain Reaction

    33) Product Images from "Optimized design of antisense oligomers for targeted rRNA depletion"

    Article Title: Optimized design of antisense oligomers for targeted rRNA depletion

    Journal: bioRxiv

    doi: 10.1101/2020.06.24.169102

    a) Schematic of rRNA depletion strategy using 39-40nt antisense oligos spaced ≤ 30-nt apart. b) X. laevis stage 0 total RNA (input, lane 1) and with rRNA depletion using different reaction conditions visualized on a 1% formaldehyde 1.2% agarose gel. In vitro transcribed mCherry mRNA was spiked into the input RNA prior to digestion. c) qRT-PCR comparing 28S rRNA levels in X. laevis stage 0 total RNA (input, left) versus depletion conditions normalized to mCherry. P values are from two-tailed paired t tests comparing depleted samples to their corresponding total RNA input. d) qRT-PCR measuring mCherry-normalized rRNA and mRNA levels in X. laevis stage 0 rRNA-depleted samples divided by levels in untreated samples. P values are from two-tailed paired t tests for each gene comparing depleted samples to their corresponding total RNA input. N.S. = not significant.
    Figure Legend Snippet: a) Schematic of rRNA depletion strategy using 39-40nt antisense oligos spaced ≤ 30-nt apart. b) X. laevis stage 0 total RNA (input, lane 1) and with rRNA depletion using different reaction conditions visualized on a 1% formaldehyde 1.2% agarose gel. In vitro transcribed mCherry mRNA was spiked into the input RNA prior to digestion. c) qRT-PCR comparing 28S rRNA levels in X. laevis stage 0 total RNA (input, left) versus depletion conditions normalized to mCherry. P values are from two-tailed paired t tests comparing depleted samples to their corresponding total RNA input. d) qRT-PCR measuring mCherry-normalized rRNA and mRNA levels in X. laevis stage 0 rRNA-depleted samples divided by levels in untreated samples. P values are from two-tailed paired t tests for each gene comparing depleted samples to their corresponding total RNA input. N.S. = not significant.

    Techniques Used: Agarose Gel Electrophoresis, In Vitro, Quantitative RT-PCR, Two Tailed Test

    34) Product Images from "An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts"

    Article Title: An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.00583

    HLA-B intron 4 sequence variability and miR-6891 isomiR characterization . (A) There are 384 annotated HLA-B alleles with full-length sequence annotation within the ImMunoGeneTics (IMGT) database (release 3.25), with each allele represented by one of eight unique intron 4 sequence motifs. The aligned sequence motifs are provided along with their allele frequency within IMGT and polymorphic positions (highlighted in red). (B) Sequence logo plot depicting the lack of polymorphism within HSA-miR-6891-5p. (C) Sequence logo plot depicting polymorphic sites within HSA-miR-6891-3p at positions 6 and 14 of the mature miRNA.
    Figure Legend Snippet: HLA-B intron 4 sequence variability and miR-6891 isomiR characterization . (A) There are 384 annotated HLA-B alleles with full-length sequence annotation within the ImMunoGeneTics (IMGT) database (release 3.25), with each allele represented by one of eight unique intron 4 sequence motifs. The aligned sequence motifs are provided along with their allele frequency within IMGT and polymorphic positions (highlighted in red). (B) Sequence logo plot depicting the lack of polymorphism within HSA-miR-6891-5p. (C) Sequence logo plot depicting polymorphic sites within HSA-miR-6891-3p at positions 6 and 14 of the mature miRNA.

    Techniques Used: Sequencing

    Predicted biogenesis of HSA-miR-6891 . miR-6891 is derived from intron 4 of HLA-B , which upon exon splicing of the HLA-B transcript forms a stable pre-miRNA hairpin structure. The pre-miRNA is then processed by the Dicer enzyme to form two mature miRNA products, HSA-miR-6891-5p and HSA-miR-6891-3p.
    Figure Legend Snippet: Predicted biogenesis of HSA-miR-6891 . miR-6891 is derived from intron 4 of HLA-B , which upon exon splicing of the HLA-B transcript forms a stable pre-miRNA hairpin structure. The pre-miRNA is then processed by the Dicer enzyme to form two mature miRNA products, HSA-miR-6891-5p and HSA-miR-6891-3p.

    Techniques Used: Derivative Assay

    Identification of potential miR-6891-5p targets . COX cells were transduced with lentiviruses expressing either antisense of HSA-miR-6891-5p or scrambled control, and altered mRNA transcript levels were assessed using microarrays. (A) Principal component analysis (PCA) was performed in order to visualize sample clustering and assess the variation among biological replicates ( N = 3 experimental and 2 controls samples). Clear circles represent the centroid of the sample clusters, and the ellipse represents 2× the standard deviation in the x - and y -axes, respectively. The first principal component accounts for 76.5% of the variance within the dataset, while the second principal component accounts for 8.6% of the variance within the dataset. (B) Hierarchical clustering of samples based upon identified differentially expressed transcripts from microarray analysis.
    Figure Legend Snippet: Identification of potential miR-6891-5p targets . COX cells were transduced with lentiviruses expressing either antisense of HSA-miR-6891-5p or scrambled control, and altered mRNA transcript levels were assessed using microarrays. (A) Principal component analysis (PCA) was performed in order to visualize sample clustering and assess the variation among biological replicates ( N = 3 experimental and 2 controls samples). Clear circles represent the centroid of the sample clusters, and the ellipse represents 2× the standard deviation in the x - and y -axes, respectively. The first principal component accounts for 76.5% of the variance within the dataset, while the second principal component accounts for 8.6% of the variance within the dataset. (B) Hierarchical clustering of samples based upon identified differentially expressed transcripts from microarray analysis.

    Techniques Used: Transduction, Expressing, Standard Deviation, Microarray

    35) Product Images from "Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors"

    Article Title: Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207948

    Molecular structures of GABA A receptors. ( A ) A cartoon representation of the major pentameric GABA A receptor subtype in the central nervous system. It contains two α1 subunits, two β2 subunits, and one γ2 subunit. This model was constructed from the cryo-EM structure (6D6U.pdb) [ 22 ] by using PYMOL. ( B ) Topology of the α1 subunit. The large N-terminal domain resides in the ER lumen or extracellular space. Ala322, displayed as a space-filling model and indicated by an arrow, is located in the third transmembrane (TM3) helix. ( C ) Sequence alignment of TM3 residues of the α1, β2, β3, and γ2 subunits of GABA A receptors. The sequences are from the following Uniprot entries: GBRA1, P14867; GBRB2, P47870-1; GBRB3, P28472-1; GBRG2, P18507-2. The A322 residue in the α1 subunit is highlighted in yellow. Hydrophobic residues are conserved in this position.
    Figure Legend Snippet: Molecular structures of GABA A receptors. ( A ) A cartoon representation of the major pentameric GABA A receptor subtype in the central nervous system. It contains two α1 subunits, two β2 subunits, and one γ2 subunit. This model was constructed from the cryo-EM structure (6D6U.pdb) [ 22 ] by using PYMOL. ( B ) Topology of the α1 subunit. The large N-terminal domain resides in the ER lumen or extracellular space. Ala322, displayed as a space-filling model and indicated by an arrow, is located in the third transmembrane (TM3) helix. ( C ) Sequence alignment of TM3 residues of the α1, β2, β3, and γ2 subunits of GABA A receptors. The sequences are from the following Uniprot entries: GBRA1, P14867; GBRB2, P47870-1; GBRB3, P28472-1; GBRG2, P18507-2. The A322 residue in the α1 subunit is highlighted in yellow. Hydrophobic residues are conserved in this position.

    Techniques Used: Construct, Sequencing

    IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: IRE1 activation increases the surface expression of α1(A322D) subunit of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or XBP1-s (spliced XBP1) plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. The cell lysates are then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B C ) (n = 5 for α1 and n = 3 for BiP, paired t-test). ( D ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface protein expression levels of α1 is shown in ( E ) (n = 5, paired t-test). ( F ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or XBP-s or transfected with XBP-s and treated with lactacystin (2.5 μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( G ) (n = 3, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Activation Assay, Expressing, Transfection, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: ATF6 activation promotes the forward trafficking of α1(A322D) subunit of GABA A receptors. (A) HEK293T cells expressing α1(A322D)β2γ2 receptors were transiently transfected with GFP or HA-tagged full-length ATF6α plasmids. Forty-eight hrs post transfection, cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis using corresponding antibodies. Endo H resistant v1 subunit bands (top arrow, lane 4) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 3 and 4) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). Quantification of total cellular protein expression levels of α1 and BiP is shown in ( B ) and ( C ) (n = 5 for α1 and n = 4 for BiP, paired t-test). Quantification of the ratio of endo H resistant α1 / total α1 is shown in ( D ) (n = 3, paired t-test). ( E ) Cells were treated as in ( A ). Forty-eight hrs post transfection, the nuclear fractions were extracted and subject to SDS-PAGE. ATF6 (N) is the cleaved, activated N-terminal ATF6 in the nucleus. Matrin-3 serves as a nuclear protein loading control. ( F ) HEK293T cells were treated as in ( A ). Forty-eight hrs post transfection, the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels is shown in ( G ) (n = 6, paired t-test). ( H ) HEK293T cells expressing α1(A322D)β2γ2 receptors were either transfected with GFP control, or ATF6, or transfected with ATF6 and treated with lactacystin (2.5μM for 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( I ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Activation Assay, Expressing, Transfection, Incubation, SDS Page, Western Blot, Affinity Purification, Quantitation Assay

    BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p
    Figure Legend Snippet: BIX, a potent BiP inducer, enhances the folding and trafficking and reduces the degradation of α1(A322D) subunits. ( A ) Chemical structure of BIX. ( B-D ) Dose response of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( B ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( C ) and ( D ) (n = 8). ( E-G ) Time course of BIX treatment in regulating α1(A322D) total protein level. HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were treated with BIX (12 μM) for the indicated time. Cells were then lysed and subjected to SDS-PAGE and Western blot analysis ( E ). Normalized band intensities for α1(A322D) subunits and BiP are shown in ( F ) and ( G ) (n = 5). ( H ) HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors were plated into a 96-well plate on day 1. Cells were then treated with BIX at the indicated concentrations or the vehicle control DMSO in the cell culture media for 24 h. One groups of HEK293T cells stably expressing α1(A322D)β2γ2 GABA A receptors are treated with thapsigargin (2 μM, 7h) as cell toxicity positive control. Resazurin (0.15mg/ml dissolved in DPBS) is added to cells 1.5 h before plate reading. Fluorescence signal at 560 nm excitation / 590 nm emission is measured. The ratios of fluorescence signal in the DMSO treatment group to treatment groups is shown in ( H ) (n = 4, one-way ANOVA). ( I ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then cells were lysed, and total proteins were extracted. Total cellular proteins were incubated with or without endoglycosidase H enzyme (endo H) or peptide-N-glycosidase F (PNGase F) for 1h at 37°C and then subjected to SDS-PAGE and Western blot analysis. Endo H resistant α1 subunit bands (top arrows, lanes 6–9) represent properly folded, post-ER α1 subunit glycoforms that traffic at least to the Golgi compartment, whereas endo H sensitive α1 subunit bands (bottom arrow, lanes 6–9) represent immature α1 subunit glycoforms that are retained in the ER. The PNGase F enzyme cleaves between the innermost N-acetyl-D-glucosamine and asparagine residues from N-linked glycoproteins, serving as a control for unglycosylated α1 subunits (lane 5). The ratio of endo H resistant α1 / total α1, which was calculated from endo H-resistant band intensity / (endo H-resistant + endo H-sensitive band intensity), serves as a measure of trafficking efficiency of the α1(A322D) subunit. Quantification of this ratio after endo H treatment (lanes 6–9) is shown in ( J ) (n = 3, paired t-test). ( K ) HEK293T cells stably expressing α1(A322D)β2γ2 receptors were either treated with DMSO vehicle control, or BIX (12 μM, 24 h) or BIX (12 μM, 24 h) and lactacystin (2.5μM, 24h). Cycloheximide (150 μg/ml), a protein synthesis inhibitor, was added to different cell groups for 0, 0.5 hr, 1 hr, and 2 hrs. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. The quantitation results are shown in ( L ) (n = 5, one-way ANOVA followed by Fisher test, *, p

    Techniques Used: Stable Transfection, Expressing, Cell Culture, SDS Page, Western Blot, Positive Control, Fluorescence, Incubation, Quantitation Assay

    BIX enhances the surface expression of α1 subunit variants of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels to the Na + /K + -ATPase controls is shown in ( B ) (n = 5, paired t-test). ( C ) HEK293T cells expressing α1β2γ2 receptors or α1(D219N)β2γ2 receptors were treated as in ( A ). Quantification of normalized total and surface WT α1 protein levels is shown in ( D F ) (n = 6 for total and n = 5 for surface, paired t-test). Quantification of normalized total and surface α1(D219N) protein levels is shown in ( E G ) (n = 6 for total and surface, paired t-test). ( H ) SH-SY5Y cells stably expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then surface biotinylation assay was performed as in ( A ). Quantification of normalized surface α1(A322D) protein levels is shown in ( I ) (n = 3, two tailed student t-test). * p
    Figure Legend Snippet: BIX enhances the surface expression of α1 subunit variants of GABA A receptors. ( A ) HEK293T cells expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then the cell surface proteins were tagged with biotin using membrane-impermeable biotinylation reagent sulfo-NHS SS-Biotin. Biotinylated surface proteins were affinity-purified using neutravidin-conjugated beads and then subjected to SDS-PAGE and Western blot analysis. The Na + /K + -ATPase serves as a surface protein loading control. Quantification of normalized surface α1(A322D) protein levels to the Na + /K + -ATPase controls is shown in ( B ) (n = 5, paired t-test). ( C ) HEK293T cells expressing α1β2γ2 receptors or α1(D219N)β2γ2 receptors were treated as in ( A ). Quantification of normalized total and surface WT α1 protein levels is shown in ( D F ) (n = 6 for total and n = 5 for surface, paired t-test). Quantification of normalized total and surface α1(D219N) protein levels is shown in ( E G ) (n = 6 for total and surface, paired t-test). ( H ) SH-SY5Y cells stably expressing α1(A322D)β2γ2 receptors were treated with BIX (12 μM, 24 h) or DMSO vehicle control. Then surface biotinylation assay was performed as in ( A ). Quantification of normalized surface α1(A322D) protein levels is shown in ( I ) (n = 3, two tailed student t-test). * p

    Techniques Used: Expressing, Affinity Purification, SDS Page, Western Blot, Stable Transfection, Surface Biotinylation Assay, Two Tailed Test

    36) Product Images from "A Novel Role of Chromodomain Protein CBX8 in DNA Damage Response *"

    Article Title: A Novel Role of Chromodomain Protein CBX8 in DNA Damage Response *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.725879

    CBX proteins are recruited to the sites of DNA damage. A , HeLa cells were plated in a 4-well chamber slide, transfected with CBX6-YFP, CBX7-YFP, or CBX8-YFP and treated with IdU. DNA was subsequently damaged using UV laser microirradiation. The cells
    Figure Legend Snippet: CBX proteins are recruited to the sites of DNA damage. A , HeLa cells were plated in a 4-well chamber slide, transfected with CBX6-YFP, CBX7-YFP, or CBX8-YFP and treated with IdU. DNA was subsequently damaged using UV laser microirradiation. The cells

    Techniques Used: Transfection

    Proteins responsible for the recruitment of CBX8 to the sites of DNA breaks. A , CBX8-YFP localization was imaged after DNA damage in HeLa cells either untreated or treated with 5 μ m PARPi (KU-0058948). B , HeLa cells treated with various concentrations
    Figure Legend Snippet: Proteins responsible for the recruitment of CBX8 to the sites of DNA breaks. A , CBX8-YFP localization was imaged after DNA damage in HeLa cells either untreated or treated with 5 μ m PARPi (KU-0058948). B , HeLa cells treated with various concentrations

    Techniques Used:

    Knockdown of CBX8 decreases the frequency of HR/NHEJ and sensitizes U2OS cells to ionizing radiation. A , DR-U2OS cells with a stably integrated tandem GFP reporter for HR were treated with pCBASce (plasmid bearing I-SceI endonuclease) or CAG (control
    Figure Legend Snippet: Knockdown of CBX8 decreases the frequency of HR/NHEJ and sensitizes U2OS cells to ionizing radiation. A , DR-U2OS cells with a stably integrated tandem GFP reporter for HR were treated with pCBASce (plasmid bearing I-SceI endonuclease) or CAG (control

    Techniques Used: Non-Homologous End Joining, Stable Transfection, Plasmid Preparation

    Structure-function analysis of the CBX8 domains responsible for its recruitment to sites of DNA damage. A , the domain architecture of CBX8 is indicated above the WT construct. The various deletion segments of CBX8 were cloned into pEGFP-N3 vector as indicated.
    Figure Legend Snippet: Structure-function analysis of the CBX8 domains responsible for its recruitment to sites of DNA damage. A , the domain architecture of CBX8 is indicated above the WT construct. The various deletion segments of CBX8 were cloned into pEGFP-N3 vector as indicated.

    Techniques Used: Construct, Clone Assay, Plasmid Preparation

    CBX8 recruitment is independent of other PRC components and PI3-like kinase pathway. A , HeLa cells were plated onto a glass-bottomed chamber slide and transfected with CBX8-YFP. The cells were treated overnight with IdU, as well as either DMSO or 10 μ
    Figure Legend Snippet: CBX8 recruitment is independent of other PRC components and PI3-like kinase pathway. A , HeLa cells were plated onto a glass-bottomed chamber slide and transfected with CBX8-YFP. The cells were treated overnight with IdU, as well as either DMSO or 10 μ

    Techniques Used: Transfection

    37) Product Images from "A novel ribosomal protein S20 variant in a family with unexplained colorectal cancer and polyposis"

    Article Title: A novel ribosomal protein S20 variant in a family with unexplained colorectal cancer and polyposis

    Journal: bioRxiv

    doi: 10.1101/2019.12.16.877084

    Confirmation of splicing defect. A) RPS20 NM_001023.3 transcript with variants linked to CRC. c.147dupA (Nieminen et al, 2014). c.177+1G > A (this study). c.181_182delTT (Broderick et al, 2017). B) RT-PCR of RPS20 from Lynch syndrome, FAP, and RPS20 c.177+1G > A subjects. FAP (familial adenomatous polyposis); NTC (no template control); NRTC (no reverse transcriptase control). C) 591bp band corresponding to partial intron 3 inclusion. Black arrow indicates c.177+1G > A variant position. Note the chromatogram shows only adenine. D) 444bp band corresponding to wild-type NM_001023.3 sequence. E) 370bp band corresponding to exon 3 skipping.
    Figure Legend Snippet: Confirmation of splicing defect. A) RPS20 NM_001023.3 transcript with variants linked to CRC. c.147dupA (Nieminen et al, 2014). c.177+1G > A (this study). c.181_182delTT (Broderick et al, 2017). B) RT-PCR of RPS20 from Lynch syndrome, FAP, and RPS20 c.177+1G > A subjects. FAP (familial adenomatous polyposis); NTC (no template control); NRTC (no reverse transcriptase control). C) 591bp band corresponding to partial intron 3 inclusion. Black arrow indicates c.177+1G > A variant position. Note the chromatogram shows only adenine. D) 444bp band corresponding to wild-type NM_001023.3 sequence. E) 370bp band corresponding to exon 3 skipping.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Variant Assay, Sequencing

    Pedigree of RPS20 NM_001023.3:c.177+1G > A family. Arrow indicates proband. Age of individual at time of study or death indicated below square/circle. Cancers are reported by type and age at diagnosis. RPS20- indicates wild type sequence. RPS20+ indicates c.177+1G > A variant. Diamonds include number of additional siblings.
    Figure Legend Snippet: Pedigree of RPS20 NM_001023.3:c.177+1G > A family. Arrow indicates proband. Age of individual at time of study or death indicated below square/circle. Cancers are reported by type and age at diagnosis. RPS20- indicates wild type sequence. RPS20+ indicates c.177+1G > A variant. Diamonds include number of additional siblings.

    Techniques Used: Sequencing, Variant Assay

    Related Articles

    DNA Extraction:

    Article Title: Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India
    Article Snippet: .. The white coloured transformants were screened by the colony PCR and one of the positive clones was grown in sufficient amount for plasmid DNA isolation by the QIAprep® Spin Miniprep Kit (Qiagen Group, Germany) and sequenced (SciGenom Labs Private Ltd, Kerala). .. The nucleotide sequence data was submitted to GenBank for accessioning.

    Clone Assay:

    Article Title: Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India
    Article Snippet: .. The white coloured transformants were screened by the colony PCR and one of the positive clones was grown in sufficient amount for plasmid DNA isolation by the QIAprep® Spin Miniprep Kit (Qiagen Group, Germany) and sequenced (SciGenom Labs Private Ltd, Kerala). .. The nucleotide sequence data was submitted to GenBank for accessioning.

    Article Title: Genome-Wide Identification of Genes Conferring Energy Related Resistance to a Synthetic Antimicrobial Peptide (Bac8c)
    Article Snippet: .. To confirm insert sizes and numbers of positive transformants, plasmids were isolated from random clones for each sized library using Qiaprep Spin MiniPrep Kit (Qiagen). .. Purified plasmids were then analyzed by either PCR using primers SL1 ( 5′-CAGTCCAGTTACGCTGGAGTC-3′ ) and SR2 ( 5′-GGTCAGGTATGATTTAAATGGTCAGT ) or by restriction digestion.

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: .. NaCl (Fisher BioReagents, cat. no. BP358-10; dissolved in H2 O at 2 M) l -Ascorbic acid (Sigma-Aldrich, cat. no. 255564; dissolved in H2 O at 40 mM) DTT (Roche Diagnostics, cat. no. 93889320; dissolved in H2 O at 50 mM) Disodium salt ATP (Fisher BioReagents, cat. no. BP413-25; dissolved in H2 O at 24 mM) α-Ketoglutaric acid disodium salt hydrate (α-KG; Sigma-Aldrich, cat. no. K3752; dissolved in H2 O at 20 mM) Tet oxidation reagent 1 (Reagent Setup) Tet oxidation reagent 2 (Reagent Setup) Micro Bio-Spin 30 columns (Bio-Rad, cat. no. 7326203) Proteinase K (Fermentas, cat. no. EO0491) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) 5-mC_test_F, 5-mC_test_R, 5-hmC_test_F, 5-hmC_test_F (synthesized by Operon; ) QIAprep spin miniprep kit (Qiagen, cat. no. 27104) Zero Blunt TOPO PCR cloning kit (Invitrogen, cat. no. K2800-20) End-It DNA end-repair kit (Epicentre, cat. no. ER81050). .. Klenow fragment (3′→5′ exo−; NEB, cat. no. M0212L) Quick ligation kit (NEB, cat. no. M2200L) TruSeq DNA sample preparation kit (Illumina, cat. no. FC-121-2001) MethylCode bisulfite conversion kit (Invitrogen, cat. no. MECOV-50) PfuTurbo Cx hotstart DNA polymerase (Agilent, cat. no. 600410) Acetonitrile (CH3 CN; Fisher Scientific, cat. no. A9984) Triethylammonium acetate, 1 M solution (TEAA; Calbiochem, cat. no. 625718) Liquid nitrogen S-Adenosylmethionine

    Selection:

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis. .. Inserts were sequenced with the assistance of the University of Alberta Department of Biological Sciences Molecular Biology Service Unit using an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA).

    Agarose Gel Electrophoresis:

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Enterococcal DNA (50-kb fragments) were purified using the Qiagen DNeasy Blood and Tissue kit (following pretreatment with lysozyme), and preparations were separated on a 1, 2, or 3% (wt/vol) agarose gel containing the DNA stain SYBRsafe (Invitrogen) exposed to a current of 300 mA for 90 min. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen), and preparations were separated on 0.9% agarose gels. .. Gels were observed in a UV light box and photographed using GeneScan software.

    Nucleic Acid Electrophoresis:

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis. .. Inserts were sequenced with the assistance of the University of Alberta Department of Biological Sciences Molecular Biology Service Unit using an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA).

    Isolation:

    Article Title: Efficient Site-Directed Saturation Mutagenesis Using Degenerate Oligonucleotides
    Article Snippet: .. For starting template, we utilized a recombinant plasmid (approximately 6.5 kb) isolated using a QIAGEN QIAprep Spin Miniprep Kit (Valencia, CA). .. This methodology requires methylated parental DNA, which is the case for most commonly utilized E. coli strains.

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. KS14 plasmid prophage DNA was isolated from five putatively lysogenized KS14-resistant C6433 isolates [ ] using a QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany). .. Lysogeny was predicted using PCR with KS14-specifc primers (KS14F: GCAGCTAACCGAGTCGCACG, KS14R: CTCTGAAAAGGTGGGCGGTGG) (Sigma-Genosys, Oakville, ON) and TopTaq DNA polymerase and buffers (Qiagen).

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis. .. Inserts were sequenced with the assistance of the University of Alberta Department of Biological Sciences Molecular Biology Service Unit using an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA).

    Article Title: Genome-Wide Identification of Genes Conferring Energy Related Resistance to a Synthetic Antimicrobial Peptide (Bac8c)
    Article Snippet: .. To confirm insert sizes and numbers of positive transformants, plasmids were isolated from random clones for each sized library using Qiaprep Spin MiniPrep Kit (Qiagen). .. Purified plasmids were then analyzed by either PCR using primers SL1 ( 5′-CAGTCCAGTTACGCTGGAGTC-3′ ) and SR2 ( 5′-GGTCAGGTATGATTTAAATGGTCAGT ) or by restriction digestion.

    Synthesized:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: .. NaCl (Fisher BioReagents, cat. no. BP358-10; dissolved in H2 O at 2 M) l -Ascorbic acid (Sigma-Aldrich, cat. no. 255564; dissolved in H2 O at 40 mM) DTT (Roche Diagnostics, cat. no. 93889320; dissolved in H2 O at 50 mM) Disodium salt ATP (Fisher BioReagents, cat. no. BP413-25; dissolved in H2 O at 24 mM) α-Ketoglutaric acid disodium salt hydrate (α-KG; Sigma-Aldrich, cat. no. K3752; dissolved in H2 O at 20 mM) Tet oxidation reagent 1 (Reagent Setup) Tet oxidation reagent 2 (Reagent Setup) Micro Bio-Spin 30 columns (Bio-Rad, cat. no. 7326203) Proteinase K (Fermentas, cat. no. EO0491) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) 5-mC_test_F, 5-mC_test_R, 5-hmC_test_F, 5-hmC_test_F (synthesized by Operon; ) QIAprep spin miniprep kit (Qiagen, cat. no. 27104) Zero Blunt TOPO PCR cloning kit (Invitrogen, cat. no. K2800-20) End-It DNA end-repair kit (Epicentre, cat. no. ER81050). .. Klenow fragment (3′→5′ exo−; NEB, cat. no. M0212L) Quick ligation kit (NEB, cat. no. M2200L) TruSeq DNA sample preparation kit (Illumina, cat. no. FC-121-2001) MethylCode bisulfite conversion kit (Invitrogen, cat. no. MECOV-50) PfuTurbo Cx hotstart DNA polymerase (Agilent, cat. no. 600410) Acetonitrile (CH3 CN; Fisher Scientific, cat. no. A9984) Triethylammonium acetate, 1 M solution (TEAA; Calbiochem, cat. no. 625718) Liquid nitrogen S-Adenosylmethionine

    Construct:

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis. .. Inserts were sequenced with the assistance of the University of Alberta Department of Biological Sciences Molecular Biology Service Unit using an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA).

    Purification:

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Enterococcal DNA (50-kb fragments) were purified using the Qiagen DNeasy Blood and Tissue kit (following pretreatment with lysozyme), and preparations were separated on a 1, 2, or 3% (wt/vol) agarose gel containing the DNA stain SYBRsafe (Invitrogen) exposed to a current of 300 mA for 90 min. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen), and preparations were separated on 0.9% agarose gels. .. Gels were observed in a UV light box and photographed using GeneScan software.

    Staining:

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Enterococcal DNA (50-kb fragments) were purified using the Qiagen DNeasy Blood and Tissue kit (following pretreatment with lysozyme), and preparations were separated on a 1, 2, or 3% (wt/vol) agarose gel containing the DNA stain SYBRsafe (Invitrogen) exposed to a current of 300 mA for 90 min. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen), and preparations were separated on 0.9% agarose gels. .. Gels were observed in a UV light box and photographed using GeneScan software.

    Polymerase Chain Reaction:

    Article Title: Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India
    Article Snippet: .. The white coloured transformants were screened by the colony PCR and one of the positive clones was grown in sufficient amount for plasmid DNA isolation by the QIAprep® Spin Miniprep Kit (Qiagen Group, Germany) and sequenced (SciGenom Labs Private Ltd, Kerala). .. The nucleotide sequence data was submitted to GenBank for accessioning.

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: .. NaCl (Fisher BioReagents, cat. no. BP358-10; dissolved in H2 O at 2 M) l -Ascorbic acid (Sigma-Aldrich, cat. no. 255564; dissolved in H2 O at 40 mM) DTT (Roche Diagnostics, cat. no. 93889320; dissolved in H2 O at 50 mM) Disodium salt ATP (Fisher BioReagents, cat. no. BP413-25; dissolved in H2 O at 24 mM) α-Ketoglutaric acid disodium salt hydrate (α-KG; Sigma-Aldrich, cat. no. K3752; dissolved in H2 O at 20 mM) Tet oxidation reagent 1 (Reagent Setup) Tet oxidation reagent 2 (Reagent Setup) Micro Bio-Spin 30 columns (Bio-Rad, cat. no. 7326203) Proteinase K (Fermentas, cat. no. EO0491) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) 5-mC_test_F, 5-mC_test_R, 5-hmC_test_F, 5-hmC_test_F (synthesized by Operon; ) QIAprep spin miniprep kit (Qiagen, cat. no. 27104) Zero Blunt TOPO PCR cloning kit (Invitrogen, cat. no. K2800-20) End-It DNA end-repair kit (Epicentre, cat. no. ER81050). .. Klenow fragment (3′→5′ exo−; NEB, cat. no. M0212L) Quick ligation kit (NEB, cat. no. M2200L) TruSeq DNA sample preparation kit (Illumina, cat. no. FC-121-2001) MethylCode bisulfite conversion kit (Invitrogen, cat. no. MECOV-50) PfuTurbo Cx hotstart DNA polymerase (Agilent, cat. no. 600410) Acetonitrile (CH3 CN; Fisher Scientific, cat. no. A9984) Triethylammonium acetate, 1 M solution (TEAA; Calbiochem, cat. no. 625718) Liquid nitrogen S-Adenosylmethionine

    Gel Extraction:

    Article Title: Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine
    Article Snippet: .. NaCl (Fisher BioReagents, cat. no. BP358-10; dissolved in H2 O at 2 M) l -Ascorbic acid (Sigma-Aldrich, cat. no. 255564; dissolved in H2 O at 40 mM) DTT (Roche Diagnostics, cat. no. 93889320; dissolved in H2 O at 50 mM) Disodium salt ATP (Fisher BioReagents, cat. no. BP413-25; dissolved in H2 O at 24 mM) α-Ketoglutaric acid disodium salt hydrate (α-KG; Sigma-Aldrich, cat. no. K3752; dissolved in H2 O at 20 mM) Tet oxidation reagent 1 (Reagent Setup) Tet oxidation reagent 2 (Reagent Setup) Micro Bio-Spin 30 columns (Bio-Rad, cat. no. 7326203) Proteinase K (Fermentas, cat. no. EO0491) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAquick nucleotide removal kit (Qiagen, cat. no. 28304) 5-mC_test_F, 5-mC_test_R, 5-hmC_test_F, 5-hmC_test_F (synthesized by Operon; ) QIAprep spin miniprep kit (Qiagen, cat. no. 27104) Zero Blunt TOPO PCR cloning kit (Invitrogen, cat. no. K2800-20) End-It DNA end-repair kit (Epicentre, cat. no. ER81050). .. Klenow fragment (3′→5′ exo−; NEB, cat. no. M0212L) Quick ligation kit (NEB, cat. no. M2200L) TruSeq DNA sample preparation kit (Illumina, cat. no. FC-121-2001) MethylCode bisulfite conversion kit (Invitrogen, cat. no. MECOV-50) PfuTurbo Cx hotstart DNA polymerase (Agilent, cat. no. 600410) Acetonitrile (CH3 CN; Fisher Scientific, cat. no. A9984) Triethylammonium acetate, 1 M solution (TEAA; Calbiochem, cat. no. 625718) Liquid nitrogen S-Adenosylmethionine

    Transformation Assay:

    Article Title: Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability
    Article Snippet: .. Plasmid DNA was extracted from the cells 24h post-transfection [QIAprep spin miniprep kit (Qiagen, Germany)], and transformed into E . coli DH5α cells. .. After plating on agar plates containing IPTG and X-Gal (Sigma-Aldrich), numbers of white and blue colonies were counted.

    Recombinant:

    Article Title: Efficient Site-Directed Saturation Mutagenesis Using Degenerate Oligonucleotides
    Article Snippet: .. For starting template, we utilized a recombinant plasmid (approximately 6.5 kb) isolated using a QIAGEN QIAprep Spin Miniprep Kit (Valencia, CA). .. This methodology requires methylated parental DNA, which is the case for most commonly utilized E. coli strains.

    Plasmid Preparation:

    Article Title: Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India
    Article Snippet: .. The white coloured transformants were screened by the colony PCR and one of the positive clones was grown in sufficient amount for plasmid DNA isolation by the QIAprep® Spin Miniprep Kit (Qiagen Group, Germany) and sequenced (SciGenom Labs Private Ltd, Kerala). .. The nucleotide sequence data was submitted to GenBank for accessioning.

    Article Title: Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability
    Article Snippet: .. Plasmid DNA was extracted from the cells 24h post-transfection [QIAprep spin miniprep kit (Qiagen, Germany)], and transformed into E . coli DH5α cells. .. After plating on agar plates containing IPTG and X-Gal (Sigma-Aldrich), numbers of white and blue colonies were counted.

    Article Title: Biocidal Efficacy of Copper Alloys against Pathogenic Enterococci Involves Degradation of Genomic and Plasmid DNAs ▿
    Article Snippet: .. Enterococcal DNA (50-kb fragments) were purified using the Qiagen DNeasy Blood and Tissue kit (following pretreatment with lysozyme), and preparations were separated on a 1, 2, or 3% (wt/vol) agarose gel containing the DNA stain SYBRsafe (Invitrogen) exposed to a current of 300 mA for 90 min. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen), and preparations were separated on 0.9% agarose gels. .. Gels were observed in a UV light box and photographed using GeneScan software.

    Article Title: Efficient Site-Directed Saturation Mutagenesis Using Degenerate Oligonucleotides
    Article Snippet: .. For starting template, we utilized a recombinant plasmid (approximately 6.5 kb) isolated using a QIAGEN QIAprep Spin Miniprep Kit (Valencia, CA). .. This methodology requires methylated parental DNA, which is the case for most commonly utilized E. coli strains.

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: .. KS14 plasmid prophage DNA was isolated from five putatively lysogenized KS14-resistant C6433 isolates [ ] using a QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany). .. Lysogeny was predicted using PCR with KS14-specifc primers (KS14F: GCAGCTAACCGAGTCGCACG, KS14R: CTCTGAAAAGGTGGGCGGTGG) (Sigma-Genosys, Oakville, ON) and TopTaq DNA polymerase and buffers (Qiagen).

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    Cloning of V L and V H domain sequences from hybridomas into the R-mAb expression plasmid. ( A ) Agarose gel analysis of V L and V H domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L fragments joined by fusion PCR (F-PCR) to the <t>P1316-derived</t> joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with <t>NotI</t> and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.
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    Cloning of V L and V H domain sequences from hybridomas into the R-mAb expression plasmid. ( A ) Agarose gel analysis of V L and V H domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/eLife.43322

    Figure Lengend Snippet: Cloning of V L and V H domain sequences from hybridomas into the R-mAb expression plasmid. ( A ) Agarose gel analysis of V L and V H domain PCR products amplified from cDNA synthesized from RNA extracted from the N59/36 (anti-NR2B/GRIN2B) and K39/25 (anti-Kv2.1/KCNB1) hybridomas. The expected size of mouse IgG V L and V H domains is ≈360 bp. ( B ) Agarose gel analysis of V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316-derived joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the N59/36 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of N59/36 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp. ( E ) Agarose gel analysis of PCR products of V L domain cDNA synthesized from RNA extracted from mouse splenocytes, the fusion partner Sp2/0-Ag14, and various hybridomas after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The intact V L domains are ≈360 bp, and the digested aberrant light chains ≈180 bp.

    Article Snippet: The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Synthesized, Derivative Assay

    Cloning of anti-Kv2.1 D3/71 V L and V H domain cDNAs from a nonviable hybridoma. ( A ) Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈360 bp, and of the cleaved aberrant V L domain is ≈180 bp. ( B ) Agarose gel analysis of D3/71 V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316 joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp.

    Journal: eLife

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

    doi: 10.7554/eLife.43322

    Figure Lengend Snippet: Cloning of anti-Kv2.1 D3/71 V L and V H domain cDNAs from a nonviable hybridoma. ( A ) Agarose gel analysis of PCR amplified V L and V H domains from cDNA synthesized from RNA extracted from the non-viable D3/71 hybridoma. The panel to the right shows the V L after digestion with the BciVI restriction enzyme to cleave the Sp2/0-Ag14-derived aberrant light chain product. The expected size of mouse IgG V L and V H domains is ≈360 bp, and of the cleaved aberrant V L domain is ≈180 bp. ( B ) Agarose gel analysis of D3/71 V H and digested V L fragments joined by fusion PCR (F-PCR) to the P1316 joining fragment to create a dual IgG chain cassette. ( C ) Agarose gel analysis of colony PCR samples of transformants from the of D3/71 R-mAb project. ( D ) Agarose gel analysis of products of restriction enzyme digestion of D3/71 plasmid DNA with NotI and AscI. The plasmid backbone is seven kbp, and the intact insert comprising the V L and V H domains and the intervening joining fragment is 2.4 kbp.

    Article Snippet: The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706).

    Techniques: Clone Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Synthesized, Derivative Assay, Plasmid Preparation

    Development of pACYC-GFP. A. Constitutive promoter sequences tested during construction of pACYC-GFP. Promoters 1–4 are variants of tac promoter that reduce the promoter strength. Promoter 1 contains an extra base pair between the −10 and −35 sites. Promoters 2, 3 and 4 contain one, two or three variations in the −35 site, respectively. Variations between promoters are underlined. Gaps in aligned promoter sequences are represented with spaces. B. Flow cytometry histograms for the five promoters tested. p1-p4: promoters 1–4. C. Competition assays between cells harboring pACYCDuet-1 (GFP−) and pACYC-GFP (GFP+). The population distribution remains the same after 24 h of growth, indicating that pACYC-GFP does not affect growth rate of the cells.

    Journal: Methods in enzymology

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    doi: 10.1016/bs.mie.2018.10.027

    Figure Lengend Snippet: Development of pACYC-GFP. A. Constitutive promoter sequences tested during construction of pACYC-GFP. Promoters 1–4 are variants of tac promoter that reduce the promoter strength. Promoter 1 contains an extra base pair between the −10 and −35 sites. Promoters 2, 3 and 4 contain one, two or three variations in the −35 site, respectively. Variations between promoters are underlined. Gaps in aligned promoter sequences are represented with spaces. B. Flow cytometry histograms for the five promoters tested. p1-p4: promoters 1–4. C. Competition assays between cells harboring pACYCDuet-1 (GFP−) and pACYC-GFP (GFP+). The population distribution remains the same after 24 h of growth, indicating that pACYC-GFP does not affect growth rate of the cells.

    Article Snippet: T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega)

    Techniques: Flow Cytometry, Cytometry

    Measurement of Cas9 cleavage using GFP reporter assay. A. Schematic of cas9 and gRNA expression plasmids. The cas9 gene and sgRNA are both expressed from arabinose inducible pBAD promoters. B. Target inserted into pACYC-GFP for this study. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. EcoRI and NotI overhangs are labeled. Positions of G3A or G7T (non-target strand sequence) mutations are indicated. C. Plasmid loss assay for Cas9 targets containing a perfect sequence or seed mismatches at the third or seventh position. Empty pACYC-GFP (no target) was used as a control to ensure that the plasmid is stable in the absence of CRISPR interference. Plasmid loss (%) is the percentage of GFP- cells based on flow cytometry measurements at each time point. The average plasmid loss from 3 replicates is shown, with error representing standard deviation.

    Journal: Methods in enzymology

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    doi: 10.1016/bs.mie.2018.10.027

    Figure Lengend Snippet: Measurement of Cas9 cleavage using GFP reporter assay. A. Schematic of cas9 and gRNA expression plasmids. The cas9 gene and sgRNA are both expressed from arabinose inducible pBAD promoters. B. Target inserted into pACYC-GFP for this study. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. EcoRI and NotI overhangs are labeled. Positions of G3A or G7T (non-target strand sequence) mutations are indicated. C. Plasmid loss assay for Cas9 targets containing a perfect sequence or seed mismatches at the third or seventh position. Empty pACYC-GFP (no target) was used as a control to ensure that the plasmid is stable in the absence of CRISPR interference. Plasmid loss (%) is the percentage of GFP- cells based on flow cytometry measurements at each time point. The average plasmid loss from 3 replicates is shown, with error representing standard deviation.

    Article Snippet: T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega)

    Techniques: Reporter Assay, Expressing, Labeling, Sequencing, Plasmid Preparation, CRISPR, Flow Cytometry, Cytometry, Standard Deviation

    Design of fluorescence-based CRISPR interference assay. A. Schematic of Cas-crRNA effector complex bound to dsDNA. The spacer-protospacer RNA:DNA hybrid is shown in yellow. The PAM is highlighted in red. The PAM-proximal seed region is labeled. B. Schematic of pACYC-GFP plasmid. MCS1: multiple cloning site 1. C. Close-up schematic of GFP expression cassette in pACYC-GFP. The locations of the constitutive promoter and ssrA degradation tag are highlighted. D. Basis for fluorescence-based plasmid loss assay. As the plasmid concentration decreases due to CRISPR-based plasmid loss, the cells become less fluorescent.

    Journal: Methods in enzymology

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    doi: 10.1016/bs.mie.2018.10.027

    Figure Lengend Snippet: Design of fluorescence-based CRISPR interference assay. A. Schematic of Cas-crRNA effector complex bound to dsDNA. The spacer-protospacer RNA:DNA hybrid is shown in yellow. The PAM is highlighted in red. The PAM-proximal seed region is labeled. B. Schematic of pACYC-GFP plasmid. MCS1: multiple cloning site 1. C. Close-up schematic of GFP expression cassette in pACYC-GFP. The locations of the constitutive promoter and ssrA degradation tag are highlighted. D. Basis for fluorescence-based plasmid loss assay. As the plasmid concentration decreases due to CRISPR-based plasmid loss, the cells become less fluorescent.

    Article Snippet: T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega)

    Techniques: Fluorescence, CRISPR, Labeling, Plasmid Preparation, Clone Assay, Expressing, Concentration Assay

    Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Journal: Methods in enzymology

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems

    doi: 10.1016/bs.mie.2018.10.027

    Figure Lengend Snippet: Detecting CRISPR interference in bacterial colonies. A. Design of target sequence inserted into pACYC-GFP. The perfect target is shown, similar oligonucleotides bearing G1C, A4G, AAA PAM or AGA PAM (non-target strand sequences) mutations were used for mutant target sequences. Positions of seed mutations are indicated. The target-strand protospacer is highlighted in yellow, the seed in blue, and the PAM in red. NcoI and NotI overhangs are labeled. B. Typhoon scanned plates for perfect target, empty pACYC-GFP lacking a CRISPR target, and the four mutant target plasmids. C. Box plot of quantified intensities for colonies on each plate. The mean intensity for each colony was normalized against the average mean intensity for colonies from the empty pACYC-GFP plate ([mean intensity induced colony]/[average mean intensity for all empty pACYC-GFP colonies]). Boxes depict variation from 25 th to 75 th percentile with the line within the box representing the median value and the X marking the mean. Error bars depict the local minimum and maximum, outliers are shown as circles.

    Article Snippet: T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega)

    Techniques: CRISPR, Sequencing, Mutagenesis, Labeling

    a) Schematic of rRNA depletion strategy using 39-40nt antisense oligos spaced ≤ 30-nt apart. b) X. laevis stage 0 total RNA (input, lane 1) and with rRNA depletion using different reaction conditions visualized on a 1% formaldehyde 1.2% agarose gel. In vitro transcribed mCherry mRNA was spiked into the input RNA prior to digestion. c) qRT-PCR comparing 28S rRNA levels in X. laevis stage 0 total RNA (input, left) versus depletion conditions normalized to mCherry. P values are from two-tailed paired t tests comparing depleted samples to their corresponding total RNA input. d) qRT-PCR measuring mCherry-normalized rRNA and mRNA levels in X. laevis stage 0 rRNA-depleted samples divided by levels in untreated samples. P values are from two-tailed paired t tests for each gene comparing depleted samples to their corresponding total RNA input. N.S. = not significant.

    Journal: bioRxiv

    Article Title: Optimized design of antisense oligomers for targeted rRNA depletion

    doi: 10.1101/2020.06.24.169102

    Figure Lengend Snippet: a) Schematic of rRNA depletion strategy using 39-40nt antisense oligos spaced ≤ 30-nt apart. b) X. laevis stage 0 total RNA (input, lane 1) and with rRNA depletion using different reaction conditions visualized on a 1% formaldehyde 1.2% agarose gel. In vitro transcribed mCherry mRNA was spiked into the input RNA prior to digestion. c) qRT-PCR comparing 28S rRNA levels in X. laevis stage 0 total RNA (input, left) versus depletion conditions normalized to mCherry. P values are from two-tailed paired t tests comparing depleted samples to their corresponding total RNA input. d) qRT-PCR measuring mCherry-normalized rRNA and mRNA levels in X. laevis stage 0 rRNA-depleted samples divided by levels in untreated samples. P values are from two-tailed paired t tests for each gene comparing depleted samples to their corresponding total RNA input. N.S. = not significant.

    Article Snippet: Initial samples for the X. laevis 28S qRT-PCR were column purified (Qiagen #28704) and used at full concentration for qRT-PCR; subsequent samples were used directly at 1:10 dilution for qRT-PCR based on the results of a 4-sample, 1:5 dilution calibration curve analysis. qRT-PCR was performed in triplicate using 10μl reactions (2.5μl of cDNA, 5 μM of each forward and reverse primers, and 2x SYGreen mix (Genesee #17-505B)). qPCR was performed on QuantStudio 3 (Applied Biosystems) with an initial heat activation at 50°C for 2 minutes and then 95°C for 10 minutes.

    Techniques: Agarose Gel Electrophoresis, In Vitro, Quantitative RT-PCR, Two Tailed Test