input dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher input dna
    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) <t>DNA</t> agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) <t>Agilent</t> Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score
    Input Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue"

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2016.03.1048

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score
    Figure Legend Snippet: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Fluorescence, FACS, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Ligation

    2) Product Images from "Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats"

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgp304

    Rassf1a promoter methylation in mammary glands of control rats and rats continuously exposed to E 2 for 6 and 12 weeks. ( A ) MSP analysis of the Rassf1a first exon methylation. Bisulfite-modified DNA was PCR amplified with two sets of primers specific to
    Figure Legend Snippet: Rassf1a promoter methylation in mammary glands of control rats and rats continuously exposed to E 2 for 6 and 12 weeks. ( A ) MSP analysis of the Rassf1a first exon methylation. Bisulfite-modified DNA was PCR amplified with two sets of primers specific to

    Techniques Used: Methylation, Modification, Polymerase Chain Reaction, Amplification

    3) Product Images from "The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency *"

    Article Title: The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.599993

    RB family protein abundance decreases during differentiation concomitant with increased engagement at target gene promoters. A , FACS analysis showing increased G 1 and reduced S phase population in differentiated ES cells (− LIF , + RA ) as compared with pluripotent ES cells (+ LIF , − RA ). B , Western blot analysis of RB, p107, and p130 in whole cell extract derived from pluripotent (− RA , lane 1 ) and differentiated (+ RA , lane 2 ) mouse ES cells. RB, p107, and p130 levels in differentiated ES cells were decreased by 69, 64, and 46%, respectively, as compared with pluripotent ES cells ( n = 2). Oct4 was analyzed as a positive control of differentiation and was substantially diminished in RA-treated ES cells ( lane 2 ). Actin and tubulin were analyzed as loading controls. Whole cell extracts from MCF7 breast adenocarcinoma cells ( lane 3 ) were analyzed as a negative control for Oct4 and as a positive control for RB, p107, and p130 detection. C , quantitative real time PCR showing relative changes in the abundance of RB, p107, and p130 mRNA transcripts upon differentiation (+ RA /− RA ). RB and p107 mRNA levels increased modestly after differentiation, whereas p130 levels were modestly reduced. Transcript levels of Nanog and Pou5f1 (Oct4) were reduced after RA treatment, as expected. D , p107 and p130 association at target promoters is stimulated during RA-induced differentiation. Chromatin immunoprecipitation assays were performed with the indicated antibodies to determine enrichment of RB, p107, and p130 on the CCNA2 and MCM10 promoters before and after RA treatment. After differentiation, CCNA2 start site ( TSS ) DNA was significantly enriched in both the p107 and p130 immunoprecipitated samples ( n = 6, p
    Figure Legend Snippet: RB family protein abundance decreases during differentiation concomitant with increased engagement at target gene promoters. A , FACS analysis showing increased G 1 and reduced S phase population in differentiated ES cells (− LIF , + RA ) as compared with pluripotent ES cells (+ LIF , − RA ). B , Western blot analysis of RB, p107, and p130 in whole cell extract derived from pluripotent (− RA , lane 1 ) and differentiated (+ RA , lane 2 ) mouse ES cells. RB, p107, and p130 levels in differentiated ES cells were decreased by 69, 64, and 46%, respectively, as compared with pluripotent ES cells ( n = 2). Oct4 was analyzed as a positive control of differentiation and was substantially diminished in RA-treated ES cells ( lane 2 ). Actin and tubulin were analyzed as loading controls. Whole cell extracts from MCF7 breast adenocarcinoma cells ( lane 3 ) were analyzed as a negative control for Oct4 and as a positive control for RB, p107, and p130 detection. C , quantitative real time PCR showing relative changes in the abundance of RB, p107, and p130 mRNA transcripts upon differentiation (+ RA /− RA ). RB and p107 mRNA levels increased modestly after differentiation, whereas p130 levels were modestly reduced. Transcript levels of Nanog and Pou5f1 (Oct4) were reduced after RA treatment, as expected. D , p107 and p130 association at target promoters is stimulated during RA-induced differentiation. Chromatin immunoprecipitation assays were performed with the indicated antibodies to determine enrichment of RB, p107, and p130 on the CCNA2 and MCM10 promoters before and after RA treatment. After differentiation, CCNA2 start site ( TSS ) DNA was significantly enriched in both the p107 and p130 immunoprecipitated samples ( n = 6, p

    Techniques Used: FACS, Western Blot, Derivative Assay, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    4) Product Images from "The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism"

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.028506

    The transcription factor SEB-1 binds in vivo to the promoters of glycogenic genes. Genomic DNA from the Δ seb-1 complemented strain (Δ seb-1 his-3 :: Pccg-1-seb1-sfgfp ) subjected or not to heat stress was immunoprecipitated with anti-GFP antibody and the immunoprecipitates (IPs) were used to analyze the binding of SEB-1 to the target genes gnn , gsn , gbn , gpn , and gdn by ChIP-qPCR. A region inside the coding sequence of the ubiquitin gene was used as a negative control for binding. Values of three replicates were used for statistical analysis (* P
    Figure Legend Snippet: The transcription factor SEB-1 binds in vivo to the promoters of glycogenic genes. Genomic DNA from the Δ seb-1 complemented strain (Δ seb-1 his-3 :: Pccg-1-seb1-sfgfp ) subjected or not to heat stress was immunoprecipitated with anti-GFP antibody and the immunoprecipitates (IPs) were used to analyze the binding of SEB-1 to the target genes gnn , gsn , gbn , gpn , and gdn by ChIP-qPCR. A region inside the coding sequence of the ubiquitin gene was used as a negative control for binding. Values of three replicates were used for statistical analysis (* P

    Techniques Used: In Vivo, Immunoprecipitation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control

    5) Product Images from "A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication"

    Article Title: A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-172

    Mutational analysis of the predicted stem-loop structure in the N-SL2 . (A) Strategic representation of RT-PCR used to detect (-) gRNA, (+) sg mRNA7 and (-) sg mRNA7. The positions are according to APRRSV stain (GenBank: GQ330474 ) and all primer sequences are listed in Table 1. pAS was a non-replicative control which was absence of gene ORF1a and ORF1b (1688-13118) in full-length cDNA clone. (B) Schematic representation of the mutations introduced into the N-SL2 structure. The loop was enlarged as described in Figure 1, and mutants L-LL and L-RR were generated by overlapping PCR mutagenesis. L-RL was generated by combining the right and left arm sequences of the L-LL and L-RR, respectively, such that the overall structure of N-SL2 was restored. All the mutated nucleotides (lowercase) are highlighted in gray shading. The stem mutants, S-LL and S-RR, were generated by overlapping PCR such that one arm sequence was replaced with that of the opposite arm. The double mutant, S-RL, was generated by combining the mutations in the left and right arms such that the overall structure was restored. All mutant sequences are shown as lowercase. (C) RT-PCR of RNAs extracted from pAS and WT transfected cells at 24 hours after transfection. DNase I and RNase A were used to omit template DNA and the reverse transcriptase. The primers were nested RT-PCR primers as same as (-) gRNA detection. A 2-kbp ladder was used as a molecular size marker. The numbers indicated the lane No. (D) RT-PCR analysis of the mutants. Total cellular RNAs were extracted from mutant plasmids-transfected from BHK-21 cells at 24 hours post-transfection. β-actin is a marker for the level of intracellular RNA isolation, and pAS is a non-replicative control.
    Figure Legend Snippet: Mutational analysis of the predicted stem-loop structure in the N-SL2 . (A) Strategic representation of RT-PCR used to detect (-) gRNA, (+) sg mRNA7 and (-) sg mRNA7. The positions are according to APRRSV stain (GenBank: GQ330474 ) and all primer sequences are listed in Table 1. pAS was a non-replicative control which was absence of gene ORF1a and ORF1b (1688-13118) in full-length cDNA clone. (B) Schematic representation of the mutations introduced into the N-SL2 structure. The loop was enlarged as described in Figure 1, and mutants L-LL and L-RR were generated by overlapping PCR mutagenesis. L-RL was generated by combining the right and left arm sequences of the L-LL and L-RR, respectively, such that the overall structure of N-SL2 was restored. All the mutated nucleotides (lowercase) are highlighted in gray shading. The stem mutants, S-LL and S-RR, were generated by overlapping PCR such that one arm sequence was replaced with that of the opposite arm. The double mutant, S-RL, was generated by combining the mutations in the left and right arms such that the overall structure was restored. All mutant sequences are shown as lowercase. (C) RT-PCR of RNAs extracted from pAS and WT transfected cells at 24 hours after transfection. DNase I and RNase A were used to omit template DNA and the reverse transcriptase. The primers were nested RT-PCR primers as same as (-) gRNA detection. A 2-kbp ladder was used as a molecular size marker. The numbers indicated the lane No. (D) RT-PCR analysis of the mutants. Total cellular RNAs were extracted from mutant plasmids-transfected from BHK-21 cells at 24 hours post-transfection. β-actin is a marker for the level of intracellular RNA isolation, and pAS is a non-replicative control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, Generated, Polymerase Chain Reaction, Mutagenesis, Sequencing, Transfection, Marker, Isolation

    6) Product Images from "BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation"

    Article Title: BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation

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

    doi: 10.1073/pnas.1607687113

    ChIP assays showing that BBX21 is not associated with the HYH promoter in vivo. ChIP was performed with anti-myc antibodies, and ChIP DNA was analyzed by real-time qPCR. Error bars represent SD of three technical replicates.
    Figure Legend Snippet: ChIP assays showing that BBX21 is not associated with the HYH promoter in vivo. ChIP was performed with anti-myc antibodies, and ChIP DNA was analyzed by real-time qPCR. Error bars represent SD of three technical replicates.

    Techniques Used: Chromatin Immunoprecipitation, In Vivo, Real-time Polymerase Chain Reaction

    7) Product Images from "BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer"

    Article Title: BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer

    Journal: International Journal of Biological Sciences

    doi:

    Cister search results and PCR amplification of ChIP DNA samples from MCF7 and Hs578T cells. A. Cister predicts regulatory regions in DNA sequences by searching for clusters of cis-elements. The red line adjacent to the pink protein coding region corresponds to the putative BP1 binding site located on the positive strand of the human BRCA1 gene and indicates the probability that a regulatory factor binds at this position. B. ChIP assays were conducted with a BP1 antibody and a no antibody control. A 186 base pair PCR fragment was amplified with BP1 immunoprecipitated and input DNA from BP1 overexpressing MCF7 and Hs578T cells, but not with the no antibody control, using primers flanking the putative BP1 binding site. C. Correlation of BP1 mRNA expression with the level of ChIPed BRCA1 genomic DNA analysis. ChIP assays were performed on a series of MCF7 cell lines with different levels of BP1 expression. Various levels of BRCA1 genomic DNA were amplified from the ChIP DNA by SYBR real-time PCR, in correlation with the mRNA expression of BP1 in each cell line.
    Figure Legend Snippet: Cister search results and PCR amplification of ChIP DNA samples from MCF7 and Hs578T cells. A. Cister predicts regulatory regions in DNA sequences by searching for clusters of cis-elements. The red line adjacent to the pink protein coding region corresponds to the putative BP1 binding site located on the positive strand of the human BRCA1 gene and indicates the probability that a regulatory factor binds at this position. B. ChIP assays were conducted with a BP1 antibody and a no antibody control. A 186 base pair PCR fragment was amplified with BP1 immunoprecipitated and input DNA from BP1 overexpressing MCF7 and Hs578T cells, but not with the no antibody control, using primers flanking the putative BP1 binding site. C. Correlation of BP1 mRNA expression with the level of ChIPed BRCA1 genomic DNA analysis. ChIP assays were performed on a series of MCF7 cell lines with different levels of BP1 expression. Various levels of BRCA1 genomic DNA were amplified from the ChIP DNA by SYBR real-time PCR, in correlation with the mRNA expression of BP1 in each cell line.

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

    8) Product Images from "The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells"

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116151

    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
    Figure Legend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Techniques Used: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.
    Figure Legend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Techniques Used: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

    9) Product Images from "Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle"

    Article Title: Histone Deacetylases Play a Major Role in the Transcriptional Regulation of the Plasmodium falciparum Life Cycle

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000737

    Apicidin induces histone hyperacetylation along promoter regions and protein expression of P. falciparum genes. A. Distribution of acetylated H4K8 and H4K5 along the 5′ flanking upstream and coding regions. CHIPs were carried out with trophozoites treated with DMSO (0.005%) and apicidin (IC90) for 1 hour and with antibodies directed against either H4K8Ac or H4K5Ac. B. Distribution of H4K8Ac and H3K4me3 along the 5′ flanking upstream and coding regions of CSP. CHIPs were carried out with schizonts treated with DMSO (0.005%) and apicidin (IC90) for 1 hour and with antibodies directed against either H4K8Ac or H3K4me3. RTQ-PCR was carried out on immunoprecipated DNA and input genomic DNA obtained from DMSO and apicidin treated cells. The log 2 ratios of H4K8Ac (black bars) and H4K5Ac/H3K4me3 (white/gray bars) were calculated by using the ΔΔCt method (Ct of apicidin-treated immunoprecipitated target gene - Ct of apicidin-treated input target gene) minus (Ct of DMSO treated immunoprecipitated target gene-Ct of DMSO treated input target gene). Black box represents PCR fragment encoding the MOE. The insert boxes show the fold increase in H4K8Ac/H4K5Ac/H3K4me3 in apicidin treated samples, obtained from ChIP-chip data analysis. C and D. Trophozoites were treated with apicidin (IC90) for 1 hour. Protein samples were analyzed by SDS-PAGE followed by immunodetection using an antibody directed against H4K8Ac and H4K5Ac (C) or CSP and EBA-175 (D). Molecular weights are shown in kDa. − and + refers to DMSO and apicidin treated cells respectively.
    Figure Legend Snippet: Apicidin induces histone hyperacetylation along promoter regions and protein expression of P. falciparum genes. A. Distribution of acetylated H4K8 and H4K5 along the 5′ flanking upstream and coding regions. CHIPs were carried out with trophozoites treated with DMSO (0.005%) and apicidin (IC90) for 1 hour and with antibodies directed against either H4K8Ac or H4K5Ac. B. Distribution of H4K8Ac and H3K4me3 along the 5′ flanking upstream and coding regions of CSP. CHIPs were carried out with schizonts treated with DMSO (0.005%) and apicidin (IC90) for 1 hour and with antibodies directed against either H4K8Ac or H3K4me3. RTQ-PCR was carried out on immunoprecipated DNA and input genomic DNA obtained from DMSO and apicidin treated cells. The log 2 ratios of H4K8Ac (black bars) and H4K5Ac/H3K4me3 (white/gray bars) were calculated by using the ΔΔCt method (Ct of apicidin-treated immunoprecipitated target gene - Ct of apicidin-treated input target gene) minus (Ct of DMSO treated immunoprecipitated target gene-Ct of DMSO treated input target gene). Black box represents PCR fragment encoding the MOE. The insert boxes show the fold increase in H4K8Ac/H4K5Ac/H3K4me3 in apicidin treated samples, obtained from ChIP-chip data analysis. C and D. Trophozoites were treated with apicidin (IC90) for 1 hour. Protein samples were analyzed by SDS-PAGE followed by immunodetection using an antibody directed against H4K8Ac and H4K5Ac (C) or CSP and EBA-175 (D). Molecular weights are shown in kDa. − and + refers to DMSO and apicidin treated cells respectively.

    Techniques Used: Expressing, Polymerase Chain Reaction, Immunoprecipitation, Chromatin Immunoprecipitation, SDS Page, Immunodetection

    10) Product Images from "Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development"

    Article Title: Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11745-5

    Within nasal processes the AP2α promoter, enhancer and coding regions are enriched for Af10-dependent H3K79me2. ( a ) Schematic representation of the mouse AP2α locus. The position of qPCR amplicons is indicated. Black box; exon, white box; intron, gray box; untranslated region. ( b ) Chromatin prepared from wild-type (WT) or Mllt10 -KO (KO) nasal processes was immunoprecipitated using anti-H3K79me2 antibody (H3K79me2) or control rabbit IgG (IgG). Precipitated DNA was subjected into qPCR. Bar graph shows recovery, expressed as percentage of input. Data are obtained from 4 independent experiments. Data are mean ± s.e. Statistical differences were assessed with Student’s t -test, and p -values are shown. GD; gene desert, ActB; β-actin.
    Figure Legend Snippet: Within nasal processes the AP2α promoter, enhancer and coding regions are enriched for Af10-dependent H3K79me2. ( a ) Schematic representation of the mouse AP2α locus. The position of qPCR amplicons is indicated. Black box; exon, white box; intron, gray box; untranslated region. ( b ) Chromatin prepared from wild-type (WT) or Mllt10 -KO (KO) nasal processes was immunoprecipitated using anti-H3K79me2 antibody (H3K79me2) or control rabbit IgG (IgG). Precipitated DNA was subjected into qPCR. Bar graph shows recovery, expressed as percentage of input. Data are obtained from 4 independent experiments. Data are mean ± s.e. Statistical differences were assessed with Student’s t -test, and p -values are shown. GD; gene desert, ActB; β-actin.

    Techniques Used: Real-time Polymerase Chain Reaction, Immunoprecipitation

    11) Product Images from "Energy homeostasis targets chromosomal reconfiguration of the humanGH1 locus"

    Article Title: Energy homeostasis targets chromosomal reconfiguration of the humanGH1 locus

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI77126

    Increased association of NCOR with the human GH1 promoter region, but not the murine Gh promoter region, in response to 3 days of HFD. Pituitary chromatin from 171hGH/CS-TG mice fed HFD or LFD for 3 days was assessed by ChIP-qPCR using anti-NCOR antibody. Binding events were calculated based on signals obtained from the immunoprecipitated/input DNA amplification, using specific PCR primers for the ( A ) human GH1 and ( B ) murine Gh promoter regions as well as a control untranscribed region of mouse chromosome 6 ( Untr6 ). Results (mean ± SEM) are expressed relative to LFD group Untr6 value (arbitrarily set to 1). Significance was assessed by 1-way ANOVA with Bonferroni post-test ( n = 3). *** P
    Figure Legend Snippet: Increased association of NCOR with the human GH1 promoter region, but not the murine Gh promoter region, in response to 3 days of HFD. Pituitary chromatin from 171hGH/CS-TG mice fed HFD or LFD for 3 days was assessed by ChIP-qPCR using anti-NCOR antibody. Binding events were calculated based on signals obtained from the immunoprecipitated/input DNA amplification, using specific PCR primers for the ( A ) human GH1 and ( B ) murine Gh promoter regions as well as a control untranscribed region of mouse chromosome 6 ( Untr6 ). Results (mean ± SEM) are expressed relative to LFD group Untr6 value (arbitrarily set to 1). Significance was assessed by 1-way ANOVA with Bonferroni post-test ( n = 3). *** P

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

    12) Product Images from "Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter"

    Article Title: Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001292

    PfH2A.Z is expressed in the nucleus throughout asexual differentiation. Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1 st panel). Pre-immune serum (pI) does not show any reactivity (2 nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3 rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4 th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R = ring stage, T = trophozoite stage, S = schizont stage. DIC = differential interference contrast.
    Figure Legend Snippet: PfH2A.Z is expressed in the nucleus throughout asexual differentiation. Full length PfH2A.Z was expressed as a GST-fusion protein in E. coli and used to immunize rabbits. (A) Specificity of antisera. Parasite extracts were separated by SDS-PAGE and analysed by western blot. Anti-PfH2A.Z antiserum specifically reacted with PfH2A.Z at 18 kDa in parasite extracts and did not cross-react with H2A at 15 kDa (1 st panel). Pre-immune serum (pI) does not show any reactivity (2 nd panel). Anti-H2A antiserum specifically detects H2A migrating at 15 kDa (3 rd panel). Anti-PfH2A.Z detects human H2A.Z in BeWo cell lysate (4 th panel). (B) Anti-PfH2A.Z immunoprecipitates acetylated PfH2A.Z. Upper panel: Anti-H4K12ac antibody labels immunoprecipitated PfH2A.Z (lane 3). Anti-H4K12ac recognises an acetylated epitope present in both H4 and PfH2A.Z. Anti-H4K12ac IP (lane 1) was performed as a positive control and shows precipitation of a band corresponding to H4. No bands are apparent after IP with pI serum (lane 2). The IgG light chain (IgGLC) from the precipitating antibodies is also detected by the secondary antibody. Lower Panel: western blot reprobed with anti-PfH2A.Z confirms specificity of the immunoprecipitation. (C) Western blot analysis across the asexual life cycle demonstrates expression of PfH2A.Z and H3 in all stages. In comparison to H3, PfH2A.Z protein expression peaks in parasites 34–40 hours post-invasion which corresponds to late trophozoites/early schizonts. The ratio of H2A.Z/H3 signal in the western blot was determined by densitometry and is presented in a bar graph. (D) Nuclear localization of PfH2A.Z is shown by indirect immunofluorescence analysis and confocal microscopy of fixed 3D7 parasites using anti-PfH2A.Z antibodies. DNA was visualized with DAPI. R = ring stage, T = trophozoite stage, S = schizont stage. DIC = differential interference contrast.

    Techniques Used: SDS Page, Western Blot, Immunoprecipitation, Positive Control, Expressing, Immunofluorescence, Confocal Microscopy

    13) Product Images from "Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue"

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2016.03.1048

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score
    Figure Legend Snippet: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Fluorescence, FACS, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Ligation

    14) Product Images from "A critical role of telomere chromatin compaction in ALT tumor cell growth"

    Article Title: A critical role of telomere chromatin compaction in ALT tumor cell growth

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa224

    HP1BP3 can localize to telomeres in both telomerase-positive and ALT cells. ( A ) 293T cells transiently co-expressing Flag-HP1BP3 together with GST-tagged TRF1 or TRF2 were harvested for co-immunoprecipitation experiments using an anti-Flag antibody. The IP products were resolved by SDS-PAGE and western blotted as indicated (Up). MBP-HP1BP3 protein was purified in vitro . Pull down assay was carried out to detect HP1BP3 interacted with SFB tagged TRF2 and TRF2 ΔTRFH from cells expressing lysis. Then WB analysis was detected using Flag and HP1BP3 antibodies (Down). ( B ) U2OS cells stably expressing vector alone, Flag-HP1BP3, or Flag-HP1 were used in telomere ChIP assays with anti-IgG, anti-Flag and anti-HP1BP3 antibodies. The precipitated DNA was slot-blotted and probed with a biotinylated telomere probe (5′-TTAGGG-3′) 3 . An ALU probe was used to control for input and IgG served as a negative control. ( C ) Signals from (B) were quantified and normalized either to input. Error bars represent mean±SD; n = 3 independent experiment. *** P
    Figure Legend Snippet: HP1BP3 can localize to telomeres in both telomerase-positive and ALT cells. ( A ) 293T cells transiently co-expressing Flag-HP1BP3 together with GST-tagged TRF1 or TRF2 were harvested for co-immunoprecipitation experiments using an anti-Flag antibody. The IP products were resolved by SDS-PAGE and western blotted as indicated (Up). MBP-HP1BP3 protein was purified in vitro . Pull down assay was carried out to detect HP1BP3 interacted with SFB tagged TRF2 and TRF2 ΔTRFH from cells expressing lysis. Then WB analysis was detected using Flag and HP1BP3 antibodies (Down). ( B ) U2OS cells stably expressing vector alone, Flag-HP1BP3, or Flag-HP1 were used in telomere ChIP assays with anti-IgG, anti-Flag and anti-HP1BP3 antibodies. The precipitated DNA was slot-blotted and probed with a biotinylated telomere probe (5′-TTAGGG-3′) 3 . An ALU probe was used to control for input and IgG served as a negative control. ( C ) Signals from (B) were quantified and normalized either to input. Error bars represent mean±SD; n = 3 independent experiment. *** P

    Techniques Used: Expressing, Immunoprecipitation, SDS Page, Western Blot, Purification, In Vitro, Pull Down Assay, Lysis, Stable Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Negative Control

    HP1BP3 knockdown or knockout increases the accessibility of telomere chromatin to micrococcal nuclease (MNase). ( A–C ) WI38VA13 cells treated with control and two HP1BP3 siRNAs were lysed and incubated with MNase (5 U) for the indicated amount of time, followed by DNA extraction and southern blot analysis using biotin-labeled telomere and ALU repeat probes. ( D ) Quantitative statistics were obtained by combining the signals in (B, C). The line chart displayed the percentage of telomeric single telomere nucleosomes of HP1BP3 KD vs. siNC single telomere nucleosomes. ** P
    Figure Legend Snippet: HP1BP3 knockdown or knockout increases the accessibility of telomere chromatin to micrococcal nuclease (MNase). ( A–C ) WI38VA13 cells treated with control and two HP1BP3 siRNAs were lysed and incubated with MNase (5 U) for the indicated amount of time, followed by DNA extraction and southern blot analysis using biotin-labeled telomere and ALU repeat probes. ( D ) Quantitative statistics were obtained by combining the signals in (B, C). The line chart displayed the percentage of telomeric single telomere nucleosomes of HP1BP3 KD vs. siNC single telomere nucleosomes. ** P

    Techniques Used: Knock-Out, Incubation, DNA Extraction, Southern Blot, Labeling

    HP1BP3 regulates H3K9me3 occupancy at the telomere chromatin. ( A ) U2OS cells stably expressing vector alone or Flag-tagged HP1BP3 were western blotted as indicated. ( B ) Cells from (A) were used for telomere ChIP analysis with the indicated antibodies. The precipitated DNA was slot blotted and probed with biotin-labeled telomere and ALU probes. Rabbit IgG served as a negative control. ( C, D ) Signals from (B) were quantified and normalized either to input (C) or histone H3 signals (D). Error bars represent mean ± SD; n = 2 independent experiment * P
    Figure Legend Snippet: HP1BP3 regulates H3K9me3 occupancy at the telomere chromatin. ( A ) U2OS cells stably expressing vector alone or Flag-tagged HP1BP3 were western blotted as indicated. ( B ) Cells from (A) were used for telomere ChIP analysis with the indicated antibodies. The precipitated DNA was slot blotted and probed with biotin-labeled telomere and ALU probes. Rabbit IgG served as a negative control. ( C, D ) Signals from (B) were quantified and normalized either to input (C) or histone H3 signals (D). Error bars represent mean ± SD; n = 2 independent experiment * P

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation, Labeling, Negative Control

    HP1BP3 inhibition enhances ALT activity and elongates telomeres in ALT cells. ( A ) Genomic DNA (25, 50 or 100 ng) from control and siRNA-treated U2OS cells were used for the C-circle assay using a biotin-labeled telomeric probe (5′-CCCTAA-3) 3 . An ALU repeat probe served as input control. ( B ) Data from (A) were quantified (mean ± SD, n = 2 independent experiments). *** P
    Figure Legend Snippet: HP1BP3 inhibition enhances ALT activity and elongates telomeres in ALT cells. ( A ) Genomic DNA (25, 50 or 100 ng) from control and siRNA-treated U2OS cells were used for the C-circle assay using a biotin-labeled telomeric probe (5′-CCCTAA-3) 3 . An ALU repeat probe served as input control. ( B ) Data from (A) were quantified (mean ± SD, n = 2 independent experiments). *** P

    Techniques Used: Inhibition, Activity Assay, Labeling

    15) Product Images from "Nitrogen depletion in the fission yeast Schizosaccharomyces pombe causes nucleosome loss in both promoters and coding regions of activated genes"

    Article Title: Nitrogen depletion in the fission yeast Schizosaccharomyces pombe causes nucleosome loss in both promoters and coding regions of activated genes

    Journal: Genome Research

    doi: 10.1101/gr.098558.109

    Chromatin profiles over selected regions. The histone H3 binding to the DNA before and after nitrogen depletion is shown. ( A ) The cluster in the Tel1R region. ( B ) The region including the urg1 + , urg2 + , and urg3 + genes. Individual genes are shown in red
    Figure Legend Snippet: Chromatin profiles over selected regions. The histone H3 binding to the DNA before and after nitrogen depletion is shown. ( A ) The cluster in the Tel1R region. ( B ) The region including the urg1 + , urg2 + , and urg3 + genes. Individual genes are shown in red

    Techniques Used: Binding Assay

    16) Product Images from "Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus"

    Article Title: Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

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

    doi: 10.1073/pnas.1524857113

    Association of Dnmt3a, Dnmt1, and Tet1 with c-Fos and Egr-1 gene loci after FS. Rats were killed under baseline conditions or at 60 min after the start of a 15-min FS session (FS60). ChIP for Dnmt3a, Dnmt3b, and Tet1 was conducted on hippocampus tissue, followed by quantitative PCR (qPCR) for the c-Fos ( A ) and Egr-1 ( B ) loci studied for DNA methylation changes after SAM and FS. Data are expressed as the enrichment of the respective enzymes at the loci at FS60 relative to the enrichment in the baseline situation (mean ± SEM, n = 4). * P
    Figure Legend Snippet: Association of Dnmt3a, Dnmt1, and Tet1 with c-Fos and Egr-1 gene loci after FS. Rats were killed under baseline conditions or at 60 min after the start of a 15-min FS session (FS60). ChIP for Dnmt3a, Dnmt3b, and Tet1 was conducted on hippocampus tissue, followed by quantitative PCR (qPCR) for the c-Fos ( A ) and Egr-1 ( B ) loci studied for DNA methylation changes after SAM and FS. Data are expressed as the enrichment of the respective enzymes at the loci at FS60 relative to the enrichment in the baseline situation (mean ± SEM, n = 4). * P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, DNA Methylation Assay

    17) Product Images from "Sulforaphane Enhances Nrf2 Expression in Prostate Cancer TRAMP C1 Cells through Epigenetic Regulation"

    Article Title: Sulforaphane Enhances Nrf2 Expression in Prostate Cancer TRAMP C1 Cells through Epigenetic Regulation

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2013.02.010

    Effect of SFN on methylation of Nrf2 promoter region in TRAMP C1 cells using methylation DNA immunopredipitation (MeDIP) assay. Genomic DNA (10 μg) extracted from SFN or 5-aza/TSA treated TRAMP C1 cells were used for MeDIP analysis. Genomic DNA
    Figure Legend Snippet: Effect of SFN on methylation of Nrf2 promoter region in TRAMP C1 cells using methylation DNA immunopredipitation (MeDIP) assay. Genomic DNA (10 μg) extracted from SFN or 5-aza/TSA treated TRAMP C1 cells were used for MeDIP analysis. Genomic DNA

    Techniques Used: Methylation, Methylated DNA Immunoprecipitation

    18) Product Images from "Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease"

    Article Title: Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease

    Journal: Journal of Alzheimer's disease : JAD

    doi: 10.3233/JAD-170342

    Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Increased α-Syn expression causes DNA breaks in neurons synergistically with pro-oxidant metals. A, B) Alkaline Comet assay in iFLAG-α-Syn SHSY-5Y cells exposed to 200 μM FeSO 4 or CuSO 4 . α-Syn was induced with Dox for 48 h. Metal salts alone at the same concentration caused only moderate increases in strand breaks. C, D) Semi-quantitative LA-PCR assay for genomic DNA isolated from pCW-iFLAG-α-Syn SHSY-5Y cells in the presence of FeSO 4 or CuSO 4 . *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Expressing, Alkaline Single Cell Gel Electrophoresis, Concentration Assay, Polymerase Chain Reaction, Isolation

    Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.
    Figure Legend Snippet: Nuclear localization and chromatin/DNA binding of α-Syn. A, B) Characterization of time-dependent induction of FLAG-α-Syn in a SHSY-5Y cell line stably harboring tet-on (Dox-inducible) pCW-iFLAG-α-Syn vector. Immunoblotting (A) and immunofluorescence (B) revealed a time-dependent increase (2–4-fold) in both FLAG and total α-Syn levels after induction with Dox for 24–72 h in differentiated cells. The presence of nuclear α-Syn after 72 h of Dox induction is indicated in the enlarged image. C) PLA of FLAG versus α-Syn antibody in pCW-iFLAG-α-Syn SHSY-5Y cells. A PLA focus in the nucleus (DAPI) detected the same molecule of ectopic α-Syn. PLA of FLAG versus histone H3 antibody confirmed interaction with H3 in the nucleus. D) ChIP assay using FLAG antibody from pCW-iFLAG-α-Syn SHSY-5Y cells after Dox induction (72 h) and real-time PCR amplification using three randomly selected primer pairs. E) In vitro biotin affinity co-elution analysis. Immunoblotting of SHSY-5Y cell nuclear extract or recombinant α-Syn co-eluted with biotin-labeled duplex DNA oligo.

    Techniques Used: Binding Assay, Stable Transfection, Plasmid Preparation, Immunofluorescence, Proximity Ligation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, In Vitro, Co-Elution Assay, Recombinant, Labeling

    DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.
    Figure Legend Snippet: DNA damage in neurons generated from normal and PD patient-derived SNCA -tri iPSC cells. A) Phase contrast image demonstrating generation of NPCs from iPSCs: (a) SNCA- tri iPSCs cultured in MEF feeder layer, (b) SNCA- tri iPSCs cultured in feeder free layer, (c) day 2, (d) day 4, (e) day 6 of neural induction for NPC derivation, (f) NPC at passage 3. The iPSC specific marker Oct4 and neural precursor markers nestin analyzed by immunoblotting (B). C) Immunofluorescence characterization with nestin and α-Syn protein expression. D) α-SYN mRNA quantitation in control versus SNCA-tri iPSC and NPC cells. E, F) LA-PCR analysis of genomic DNA isolated from control or SNCA -tri NPC cells exposed to 200 μM FeSO 4 or CuSO 4 . *** p ≤ 0.001.

    Techniques Used: Generated, Derivative Assay, Cell Culture, Marker, Immunofluorescence, Expressing, Quantitation Assay, Polymerase Chain Reaction, Isolation

    19) Product Images from "Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue"

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2016.03.1048

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score
    Figure Legend Snippet: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Fluorescence, FACS, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Ligation

    20) Product Images from "An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST"

    Article Title: An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1650-14.2014

    Rest mRNA is regulated post-transcriptionally through its 3′ UTR. A , Top, Real-time RT-PCR analysis of mRNA from PC12 cells transfected with Myc-tagged REST cDNAs and treated with CHX (50 μg/ml) or vehicle alone (DMSO). n = 3 independent experiments. Bottom, Schematic showing location of the Myc tag on Rest coding sequence (CDS) and primers (red arrows) used in the analysis. B , Yeast three-hybrid screen. Components are color-coded according to the plasmid of origin. DBD, DNA binding domain of GAL4 activator protein; RevM10, RBP; RRE, HIV-1 RNA recognition element; Rest , 3′ UTR 3.4 kb untranslated region; AD, GAL4 activation domain; GAL4 binding site, upstream activator sequence driving reporter gene activation. C , Representative colony isolate from three-hybrid screen showing that antibiotic resistance and reporter gene expression (blue color) depended on both Rest 3′ UTR and HIV-1 RRE. D , Top, WT and mutant ARE sites in Rest 3′ UTR. Middle, Schematic representation of ( Luc )- Rest 3′ UTR constructs. Bottom, RT-PCR analysis of PC12 cells transfected transiently with firefly luciferase ( Luc )- Rest 3′ UTR plasmids treated with CHX or vehicle (DMSO). n = 3 independent experiments. ARE are boxed and show A to C mutations. E , Gel mobility shift (left) and Western blot analysis (right) showing binding of ZFP36L2 protein to a biotinylated probe for the predicted ZFP36L2 binding site on Rest 3′ UTR and equivalent expression of Zfp36l2 cDNAs transfected into HEK293 cells. Left, The biotinylated oligonucleotides were incubated alone (lane 1), with cell extracts of HEK293 transfected with either empty vector (lane 2), EGFP expressing vector (lane 3), EGFP- ZPF36L2 fusion vector (lane 4), or a ZPF36L2 construct encoding a mutation in its zinc-finger, EGFP-C176S ZPF36L2 (lane 5) that precludes binding to RNA. Arrow indicates a mobility-retarded complex. Right, Transfection with EGFP control (lane 1), WT EGFP-ZFP36L2 (lane 2), and mutant EGFP-ZFP36L2-C176S (lane 3). F , Western blot analysis of PC12 cells transfected with ZFP36L 2 -MYC or GFP and treated with 50 μg CHX for indicated times. α-Tubulin (αTu), loading control. HEK293 cell lane shows migration position of endogenous human ZFP36L2 recognized by antibody to human ZFP36L2. Bottom, Quantification of three independent experiments. * p
    Figure Legend Snippet: Rest mRNA is regulated post-transcriptionally through its 3′ UTR. A , Top, Real-time RT-PCR analysis of mRNA from PC12 cells transfected with Myc-tagged REST cDNAs and treated with CHX (50 μg/ml) or vehicle alone (DMSO). n = 3 independent experiments. Bottom, Schematic showing location of the Myc tag on Rest coding sequence (CDS) and primers (red arrows) used in the analysis. B , Yeast three-hybrid screen. Components are color-coded according to the plasmid of origin. DBD, DNA binding domain of GAL4 activator protein; RevM10, RBP; RRE, HIV-1 RNA recognition element; Rest , 3′ UTR 3.4 kb untranslated region; AD, GAL4 activation domain; GAL4 binding site, upstream activator sequence driving reporter gene activation. C , Representative colony isolate from three-hybrid screen showing that antibiotic resistance and reporter gene expression (blue color) depended on both Rest 3′ UTR and HIV-1 RRE. D , Top, WT and mutant ARE sites in Rest 3′ UTR. Middle, Schematic representation of ( Luc )- Rest 3′ UTR constructs. Bottom, RT-PCR analysis of PC12 cells transfected transiently with firefly luciferase ( Luc )- Rest 3′ UTR plasmids treated with CHX or vehicle (DMSO). n = 3 independent experiments. ARE are boxed and show A to C mutations. E , Gel mobility shift (left) and Western blot analysis (right) showing binding of ZFP36L2 protein to a biotinylated probe for the predicted ZFP36L2 binding site on Rest 3′ UTR and equivalent expression of Zfp36l2 cDNAs transfected into HEK293 cells. Left, The biotinylated oligonucleotides were incubated alone (lane 1), with cell extracts of HEK293 transfected with either empty vector (lane 2), EGFP expressing vector (lane 3), EGFP- ZPF36L2 fusion vector (lane 4), or a ZPF36L2 construct encoding a mutation in its zinc-finger, EGFP-C176S ZPF36L2 (lane 5) that precludes binding to RNA. Arrow indicates a mobility-retarded complex. Right, Transfection with EGFP control (lane 1), WT EGFP-ZFP36L2 (lane 2), and mutant EGFP-ZFP36L2-C176S (lane 3). F , Western blot analysis of PC12 cells transfected with ZFP36L 2 -MYC or GFP and treated with 50 μg CHX for indicated times. α-Tubulin (αTu), loading control. HEK293 cell lane shows migration position of endogenous human ZFP36L2 recognized by antibody to human ZFP36L2. Bottom, Quantification of three independent experiments. * p

    Techniques Used: Quantitative RT-PCR, Transfection, Sequencing, Plasmid Preparation, Binding Assay, Activation Assay, Expressing, Mutagenesis, Construct, Reverse Transcription Polymerase Chain Reaction, Luciferase, Mobility Shift, Western Blot, Incubation, Migration

    21) Product Images from "Topoisomerase II binds nucleosome-free DNA and acts redundantly with topoisomerase I to enhance recruitment of RNA Pol II in budding yeast"

    Article Title: Topoisomerase II binds nucleosome-free DNA and acts redundantly with topoisomerase I to enhance recruitment of RNA Pol II in budding yeast

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

    doi: 10.1073/pnas.1106834108

    Exclusive distribution of Top2 and nucleosome occupancy . ChIP DNA of Top2 and H3 and input from wild-type cells were amplified, labeled, and hybridized to Affymetrix Tiling arrays. The average binding over 6,576 annotated genes and their adjacent 500-bp
    Figure Legend Snippet: Exclusive distribution of Top2 and nucleosome occupancy . ChIP DNA of Top2 and H3 and input from wild-type cells were amplified, labeled, and hybridized to Affymetrix Tiling arrays. The average binding over 6,576 annotated genes and their adjacent 500-bp

    Techniques Used: Chromatin Immunoprecipitation, Amplification, Labeling, Binding Assay

    22) Product Images from "Characterization of the human ?-globin downstream promoter region"

    Article Title: Characterization of the human ?-globin downstream promoter region

    Journal: Nucleic Acids Research

    doi:

    In vivo footprint analysis of a conserved E-box located in the murine adult β-globin downstream promoter region. NIH3T3L1 (lane 2) or MEL (lane 3) cells were treated with DMS. The DNA was purified from these cells and analyzed by linker ligation-mediated PCR as described in Materials and Methods. Lane 1 depicts the G sequencing ladder of the region. The position of the E-box at +60 is indicated on the right. The open circle on the left highlights a G residue that is consistently protected from cleavage by DMS in MEL cells.
    Figure Legend Snippet: In vivo footprint analysis of a conserved E-box located in the murine adult β-globin downstream promoter region. NIH3T3L1 (lane 2) or MEL (lane 3) cells were treated with DMS. The DNA was purified from these cells and analyzed by linker ligation-mediated PCR as described in Materials and Methods. Lane 1 depicts the G sequencing ladder of the region. The position of the E-box at +60 is indicated on the right. The open circle on the left highlights a G residue that is consistently protected from cleavage by DMS in MEL cells.

    Techniques Used: In Vivo, Purification, Ligation, Polymerase Chain Reaction, Sequencing

    Characterization of protein–DNA interactions in the human β-globin downstream promoter region in vivo . ( A ) Chromatin immunoprecipitation (ChIP) experiment analyzing the interaction of proteins with the murine/human β-globin gene or the HS2 5′flanking region in MEL and K562 cells in vivo . Cells were incubated in 1% formaldehyde to crosslink protein–DNA and protein–protein interactions. After sonication, the cells were lysed and the chromatin was precipitated with either no antibody (lanes 3 and 11) or with antibodies specific for USF1 (lanes 4 and 12), USF2 (lanes 5 and 13), TFII-I (lanes 6 and 14), NF-E2 (p45, lanes 7 and 15) or acetylated histone H3 (lanes 8 and 16). DNA was purified from the precipitate and analyzed by PCR for the presence of the murine or human β-globin gene (210 and 321 bp, respectively, lanes 2–8) or the murine or human HS2 5′flank (336 and 565 bp, respectively, lanes 10–16). As positive controls, the input DNA was also analyzed by PCR (lanes 2 and 10). Lanes 1 and 9 show radiolabeled 100 bp markers. ( B ) Western blot analysis of protein extracts from MEL and K562 cells. Nuclear extracts were prepared from MEL or K562 cells and electrophoresed on denaturing polyacrylamide gels. Proteins were electroblotted to a nitrocellulose membrane, which was then hybridized to antibodies specific for USF1, USF2, TFII-I or NF-E2 (p45) as indicated. Bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibody and autoradiography.
    Figure Legend Snippet: Characterization of protein–DNA interactions in the human β-globin downstream promoter region in vivo . ( A ) Chromatin immunoprecipitation (ChIP) experiment analyzing the interaction of proteins with the murine/human β-globin gene or the HS2 5′flanking region in MEL and K562 cells in vivo . Cells were incubated in 1% formaldehyde to crosslink protein–DNA and protein–protein interactions. After sonication, the cells were lysed and the chromatin was precipitated with either no antibody (lanes 3 and 11) or with antibodies specific for USF1 (lanes 4 and 12), USF2 (lanes 5 and 13), TFII-I (lanes 6 and 14), NF-E2 (p45, lanes 7 and 15) or acetylated histone H3 (lanes 8 and 16). DNA was purified from the precipitate and analyzed by PCR for the presence of the murine or human β-globin gene (210 and 321 bp, respectively, lanes 2–8) or the murine or human HS2 5′flank (336 and 565 bp, respectively, lanes 10–16). As positive controls, the input DNA was also analyzed by PCR (lanes 2 and 10). Lanes 1 and 9 show radiolabeled 100 bp markers. ( B ) Western blot analysis of protein extracts from MEL and K562 cells. Nuclear extracts were prepared from MEL or K562 cells and electrophoresed on denaturing polyacrylamide gels. Proteins were electroblotted to a nitrocellulose membrane, which was then hybridized to antibodies specific for USF1, USF2, TFII-I or NF-E2 (p45) as indicated. Bands were visualized by incubation with horseradish peroxidase-conjugated secondary antibody and autoradiography.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Incubation, Sonication, Purification, Polymerase Chain Reaction, Western Blot, Autoradiography

    23) Product Images from "High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets"

    Article Title: High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.9b00856

    Sequencing ultrashort reads on the MinION. (a) (1) Molecular inversion probes (MIPs) anneal adjacent to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). Phusion polymerase copies the target sequence into the MIP; the lack of 5′ → 3′ exonuclease activity ensures that extension halts when the polymerase reaches AS2. (2) Ampligase ligates the extended template to the phosphorylated 5′ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. (3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultralong, concatemerized ssDNA. (4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. (5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 h. (b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.
    Figure Legend Snippet: Sequencing ultrashort reads on the MinION. (a) (1) Molecular inversion probes (MIPs) anneal adjacent to the target sequence (blue) at anchor site 1 (AS1, orange) and anchor site 2 (AS2, green). Phusion polymerase copies the target sequence into the MIP; the lack of 5′ → 3′ exonuclease activity ensures that extension halts when the polymerase reaches AS2. (2) Ampligase ligates the extended template to the phosphorylated 5′ end of the MIP, generating circular ssDNA. Linear ss- or dsDNA fragments are degraded by a combination of exonuclease I and exonuclease III. (3) The circular DNA is subjected to RCA to generate tandem repeats of the original target, yielding ultralong, concatemerized ssDNA. (4) The RCA product is converted to dsDNA with Taq polymerase and subjected to ONT library preparation. (5) Sequencing reads are collected from a new MinION R9.4 flow-cell run for 24 h. (b) The raw sequences are compiled and analyzed. The identified repeats have poor accuracy in isolation, but since the sequencing errors vary across repeats, they can be aligned together to produce a high-fidelity consensus sequence.

    Techniques Used: Sequencing, Activity Assay, Flow Cytometry, Isolation

    24) Product Images from "Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes"

    Article Title: Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-12-1223

    Distributions of Spo11-FLAG and Mre11-FLAG on meiotic chromosome VI. (A) Summary of meiotic events in SK1 background strain. Rep, premeiotic DNA replication; DSB, DSB formation; Rec, homologous recombination; MI, meiosis I. (B and C) ChIP-chip analyses of Spo11-FLAG (B) and Mre11-FLAG (C). RKD1311 ( SPO11-FLAG ) and RKD1313 ( MRE11-FLAG ) cells were cross-linked at indicated time points after the medium change and analyzed. The horizontal axis represents the physical position on the chromosome. The vertical axis represents the relative binding strength of these proteins as compared with the Input signals. The scale of the vertical axis is expressed in log 2 . Black and light gray vertical bars represent detection loci showing significant binding ratios and insufficient enrichment in the immunoprecipitated fraction, respectively. The position of the centromere is indicated with the black circle on the horizontal axis. Timing of the Spo11-FLAG and Mre11-FLAG binding along chromosome VI is indicated by shaded boxes (from black to white) above the top panel. Shaded parts under the coordinate axis in B represent detection loci for the mapping (for details, see Supplemental Figure S1). Names and locations of known ARS s are shown at the bottom of each figure. Underlined ARS and light gray-colored ARS represent active early and inactive replication origins, respectively.
    Figure Legend Snippet: Distributions of Spo11-FLAG and Mre11-FLAG on meiotic chromosome VI. (A) Summary of meiotic events in SK1 background strain. Rep, premeiotic DNA replication; DSB, DSB formation; Rec, homologous recombination; MI, meiosis I. (B and C) ChIP-chip analyses of Spo11-FLAG (B) and Mre11-FLAG (C). RKD1311 ( SPO11-FLAG ) and RKD1313 ( MRE11-FLAG ) cells were cross-linked at indicated time points after the medium change and analyzed. The horizontal axis represents the physical position on the chromosome. The vertical axis represents the relative binding strength of these proteins as compared with the Input signals. The scale of the vertical axis is expressed in log 2 . Black and light gray vertical bars represent detection loci showing significant binding ratios and insufficient enrichment in the immunoprecipitated fraction, respectively. The position of the centromere is indicated with the black circle on the horizontal axis. Timing of the Spo11-FLAG and Mre11-FLAG binding along chromosome VI is indicated by shaded boxes (from black to white) above the top panel. Shaded parts under the coordinate axis in B represent detection loci for the mapping (for details, see Supplemental Figure S1). Names and locations of known ARS s are shown at the bottom of each figure. Underlined ARS and light gray-colored ARS represent active early and inactive replication origins, respectively.

    Techniques Used: Homologous Recombination, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

    25) Product Images from "Dendritic Cell-Associated miRNAs Are Modulated via Chromatin Remodeling in Response to Different Environments"

    Article Title: Dendritic Cell-Associated miRNAs Are Modulated via Chromatin Remodeling in Response to Different Environments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0090231

    Silencing of epigenetic factors affects miRNA expression and H3K4me3enrichment. ( A ) qRT-PCR analysis of miR-146a and miR-155 in moDCs transfected with various proteins. MoDCs were respectively transfected by siRNAs against MLL (siMLL), RBBP5 (siRBBP5), EZH2 (siEZH2), EED (siEED) or by mock siRNA as negative control (NC). After 48 hrs, total RNA was extracted, and miR-146a and miR-155 expression was analyzed. ( B and C ) H3K4me3 and H3K27me3 modifications at the TSS of miR-146a and miR-155. Significant histone modifications peaks were identified by CHIPOTle. The miRNA positions are predicted by Ensembl. The positions of primers used in ChIP-PCR are labeled in red. TSS, transcription start site. ( D and E ) ChIP-PCR analysis of H3K4me3 modification around the TSSs of miR-146a and miR-155. MoDCs were transfected with siMLL, siRBBP5 or NC. ChIP was performed with an anti-H3K4me3 antibody, and H3K4me3 enrichments were analyzed by qRT-PCR. Enrichments (relative to the input DNA in specific genomic regions) were assessed by ChIP. The primer sets are listed in Table S1 . The qRT-PCR data are representative of three healthy donors. The arrow points in the direction of gene transcription. R.E, relative expression. *, P
    Figure Legend Snippet: Silencing of epigenetic factors affects miRNA expression and H3K4me3enrichment. ( A ) qRT-PCR analysis of miR-146a and miR-155 in moDCs transfected with various proteins. MoDCs were respectively transfected by siRNAs against MLL (siMLL), RBBP5 (siRBBP5), EZH2 (siEZH2), EED (siEED) or by mock siRNA as negative control (NC). After 48 hrs, total RNA was extracted, and miR-146a and miR-155 expression was analyzed. ( B and C ) H3K4me3 and H3K27me3 modifications at the TSS of miR-146a and miR-155. Significant histone modifications peaks were identified by CHIPOTle. The miRNA positions are predicted by Ensembl. The positions of primers used in ChIP-PCR are labeled in red. TSS, transcription start site. ( D and E ) ChIP-PCR analysis of H3K4me3 modification around the TSSs of miR-146a and miR-155. MoDCs were transfected with siMLL, siRBBP5 or NC. ChIP was performed with an anti-H3K4me3 antibody, and H3K4me3 enrichments were analyzed by qRT-PCR. Enrichments (relative to the input DNA in specific genomic regions) were assessed by ChIP. The primer sets are listed in Table S1 . The qRT-PCR data are representative of three healthy donors. The arrow points in the direction of gene transcription. R.E, relative expression. *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Labeling, Modification

    26) Product Images from "Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib"

    Article Title: Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0152465

    ATRA decreases the binding of Nrf2 to ARE sequences in the promoter regions of HO-1, GCLM and x-CT in BTZ-treated cells. The binding of Nrf2 to ARE was evaluated by means of ChIP in HTLA-230 cells treated with 2.5 nM BTZ, 3 μM ATRA and 3 μM ATRA + 2.5 nM BTZ. Chromatin was immunoprecipitated with anti Nrf2 or with Normal IgG antibody, as indicated. PCRs were done with primers designed for the promoter regions of HO-1 E1 enhancer (in a and b, after 6 h or 24 h of cell treatment respectively), GCLM (in c, after 24 h) and x-CT (in d, after 24 h), as detailed in Material and Methods section. The amplification of pre-cleared DNA (input) has been done to perform the normalization of results. The graphs show the mean value of three independent experiments (mean±SE); *p
    Figure Legend Snippet: ATRA decreases the binding of Nrf2 to ARE sequences in the promoter regions of HO-1, GCLM and x-CT in BTZ-treated cells. The binding of Nrf2 to ARE was evaluated by means of ChIP in HTLA-230 cells treated with 2.5 nM BTZ, 3 μM ATRA and 3 μM ATRA + 2.5 nM BTZ. Chromatin was immunoprecipitated with anti Nrf2 or with Normal IgG antibody, as indicated. PCRs were done with primers designed for the promoter regions of HO-1 E1 enhancer (in a and b, after 6 h or 24 h of cell treatment respectively), GCLM (in c, after 24 h) and x-CT (in d, after 24 h), as detailed in Material and Methods section. The amplification of pre-cleared DNA (input) has been done to perform the normalization of results. The graphs show the mean value of three independent experiments (mean±SE); *p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification

    27) Product Images from "High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies"

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025181

    ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.
    Figure Legend Snippet: ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Binding Assay

    ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).
    Figure Legend Snippet: ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Techniques Used: Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Software, Negative Control

    Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.
    Figure Legend Snippet: Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Techniques Used: Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Expressing, Activity Assay, Plasmid Preparation, Transfection

    28) Product Images from "Helicobacter pylori-induced STAT3 activation and signalling network in gastric cancer"

    Article Title: Helicobacter pylori-induced STAT3 activation and signalling network in gastric cancer

    Journal: Oncoscience

    doi:

    Identification of key tumorigenic-regulators directly controlled by STAT3 following H. pylori infection (A) 849 genes were differently expressed in ATCC43504-infected AGS relative to control group. (B) Top 11 dysregulated pathways using the KEGG database. (C) Gene ontology of the cDNA microarray. (D) Quantitative RT-PCR validation of the 9 potential targets in AGS cells treated with ATCC43504 for 30 min relative to the untreated cells. GAPDH was used as an internal control. (E) Confirmation of the potential pSTAT3 target genes by ChIP-PCR using anti-pSTAT3 (Tyr705) antibody or irrelevant antibody against IgG (negative control) on ATCC43504 infected and non-infected AGS cells. Input represents the genomic DNA. * P
    Figure Legend Snippet: Identification of key tumorigenic-regulators directly controlled by STAT3 following H. pylori infection (A) 849 genes were differently expressed in ATCC43504-infected AGS relative to control group. (B) Top 11 dysregulated pathways using the KEGG database. (C) Gene ontology of the cDNA microarray. (D) Quantitative RT-PCR validation of the 9 potential targets in AGS cells treated with ATCC43504 for 30 min relative to the untreated cells. GAPDH was used as an internal control. (E) Confirmation of the potential pSTAT3 target genes by ChIP-PCR using anti-pSTAT3 (Tyr705) antibody or irrelevant antibody against IgG (negative control) on ATCC43504 infected and non-infected AGS cells. Input represents the genomic DNA. * P

    Techniques Used: Infection, Microarray, Quantitative RT-PCR, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    29) Product Images from "A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function"

    Article Title: A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku870

    Scheme of consecutive experimental steps from selective BRCA1 gene isolation in yeast S. cerevisiae to its expression in the UWB1.289 BRCA1-deficient human cells. ( a ) Step 1: A direct TAR isolation of the BRCA1 gene from human genomic DNA. TAR vector contains two gene targeting hooks (green and blue boxes), a yeast centromeric locus ( CEN ) and a yeast selectable marker HIS3. Recombination between targeting sequences in the TAR vector and the targeted sequences of the genomic DNA fragment leads to the rescue of the BRCA1 -containing loci as a circular TAR/YAC molecule. Step 2: Retrofitting of the circular TAR/YAC isolate containing the full-length BRCA1 gene by pJBRV1 vector containing a 3′ HPRT-loxP-eGFP cassette. Recombination of the Bam HI-linearized pJBRV1 vector with a TAR/YAC in yeast leads to replacement of the ColE1 origin of replication by the F’ ′ factor origin of replication that allows enable subsequent propagation in a BAC form. Step 3: BRCA1 gene loading into a unique loxP site of the alphoid tetO -HAC (tetO-HAC) by Cre-loxP recombination system in hamster CHO cells. Step 4: MMCT of alphoid tetO -HAC/BRCA1 from CHO into the human BRCA1-deficient UWB1.289 cells for complementation analyses. Step 5: Elimination of the alphoid tetO -HAC/BRCA1 from UWB1.289 cells by expression of the tTS fusion construct. ( b ) PCR analysis of the TAR clones containing the full-length BRCA1 gene for the presence of exons before and after retrofitting in yeast and after BRCA1 insertion into a loxP site of the alphoid tetO -HAC in CHO cells. The numbers above correspond to the exon number (from 1 to 24). M, ladder. ( c ) FISH analysis of the alphoid tetO -HAC/BRCA1 in CHO cells using specific probes for HAC vector (in red) and for cDNA BRCA1 gene sequences (in green). ( d ) Transcriptional analysis of the human BRCA1 gene in CHO cells. Lane 1 corresponds to a positive control: RT-PCR of RNA purified from human MCF7 cells. Lane 2 corresponds to a negative control: RT-PCR of RNA purified from CHO cells. Lanes from 3 to 6 correspond to RNA purified from five independently obtained alphoid tetO -HAC/BRCA1-containing CHO clones. Lane 7 corresponds to the RT-PCR product of the control ERCCA2 gene. Clones 3, 4, 5 and 6 are BRCA1-positive. All primers designed are presented in Supplementary Table S1. The bands have a predicted size of RT-PCR products. All amplified fragments were gel-purified and sequenced and proved the identity of products to the human BRCA1 transcripts. M, ladder. ( e ) FISH analysis of the alphoid tetO -HAC/BRCA1 in UWB1.289 cells using specific probes for HAC vector (in red). ( f ) Western blot analysis of BRCA1-deficient UWB1.289 cells, alphoid tetO -HAC/BRCA1-containing UWB1.289 cells (five independently obtained clones after MMCT transfer from the clone #4 of CHO cells) using human-specific Abs against BRCA1. BRCA1 inserted into the alphoid tetO -HAC produces a protein of the predicted size. The human breast adenocarcinoma MCF7 cell line (hemizygous for the BRCA1 wild type with a reduced BRCA1 expression) was used as a positive control for expression of BRCA1.
    Figure Legend Snippet: Scheme of consecutive experimental steps from selective BRCA1 gene isolation in yeast S. cerevisiae to its expression in the UWB1.289 BRCA1-deficient human cells. ( a ) Step 1: A direct TAR isolation of the BRCA1 gene from human genomic DNA. TAR vector contains two gene targeting hooks (green and blue boxes), a yeast centromeric locus ( CEN ) and a yeast selectable marker HIS3. Recombination between targeting sequences in the TAR vector and the targeted sequences of the genomic DNA fragment leads to the rescue of the BRCA1 -containing loci as a circular TAR/YAC molecule. Step 2: Retrofitting of the circular TAR/YAC isolate containing the full-length BRCA1 gene by pJBRV1 vector containing a 3′ HPRT-loxP-eGFP cassette. Recombination of the Bam HI-linearized pJBRV1 vector with a TAR/YAC in yeast leads to replacement of the ColE1 origin of replication by the F’ ′ factor origin of replication that allows enable subsequent propagation in a BAC form. Step 3: BRCA1 gene loading into a unique loxP site of the alphoid tetO -HAC (tetO-HAC) by Cre-loxP recombination system in hamster CHO cells. Step 4: MMCT of alphoid tetO -HAC/BRCA1 from CHO into the human BRCA1-deficient UWB1.289 cells for complementation analyses. Step 5: Elimination of the alphoid tetO -HAC/BRCA1 from UWB1.289 cells by expression of the tTS fusion construct. ( b ) PCR analysis of the TAR clones containing the full-length BRCA1 gene for the presence of exons before and after retrofitting in yeast and after BRCA1 insertion into a loxP site of the alphoid tetO -HAC in CHO cells. The numbers above correspond to the exon number (from 1 to 24). M, ladder. ( c ) FISH analysis of the alphoid tetO -HAC/BRCA1 in CHO cells using specific probes for HAC vector (in red) and for cDNA BRCA1 gene sequences (in green). ( d ) Transcriptional analysis of the human BRCA1 gene in CHO cells. Lane 1 corresponds to a positive control: RT-PCR of RNA purified from human MCF7 cells. Lane 2 corresponds to a negative control: RT-PCR of RNA purified from CHO cells. Lanes from 3 to 6 correspond to RNA purified from five independently obtained alphoid tetO -HAC/BRCA1-containing CHO clones. Lane 7 corresponds to the RT-PCR product of the control ERCCA2 gene. Clones 3, 4, 5 and 6 are BRCA1-positive. All primers designed are presented in Supplementary Table S1. The bands have a predicted size of RT-PCR products. All amplified fragments were gel-purified and sequenced and proved the identity of products to the human BRCA1 transcripts. M, ladder. ( e ) FISH analysis of the alphoid tetO -HAC/BRCA1 in UWB1.289 cells using specific probes for HAC vector (in red). ( f ) Western blot analysis of BRCA1-deficient UWB1.289 cells, alphoid tetO -HAC/BRCA1-containing UWB1.289 cells (five independently obtained clones after MMCT transfer from the clone #4 of CHO cells) using human-specific Abs against BRCA1. BRCA1 inserted into the alphoid tetO -HAC produces a protein of the predicted size. The human breast adenocarcinoma MCF7 cell line (hemizygous for the BRCA1 wild type with a reduced BRCA1 expression) was used as a positive control for expression of BRCA1.

    Techniques Used: Isolation, Expressing, Plasmid Preparation, Marker, BAC Assay, HAC Assay, Construct, Polymerase Chain Reaction, Clone Assay, Fluorescence In Situ Hybridization, Positive Control, Reverse Transcription Polymerase Chain Reaction, Purification, Negative Control, Amplification, Western Blot

    De-repression of alpha-satellite DNA transcription in BRCA1-deficient UWB1.289 cells. ( a , c and d ) Quantitative RT-PCR experiments showing that the satellite DNA transcripts of HORs regions of the chromosome 21 (chr21a and chr21b), chromosome X (chrX) ( a ), chromosome 5 (D5Z1 and D5Z2) ( d ) and DNA transcripts of pericentromeric Sat2 repeats located at 10q21 ( c ) are significantly repressed in the cells carrying alphoid tetO -HAC/BRCA1 compared to that of the cells that have lost the HAC. C t values were normalized relative to each other. Error bars indicate SD. ( b ) Timing of transcription of HORs regions of chromosome X and chromosome 21. ( e ) ChIP analysis of different centromeric regions using antibodies against CENP-B (centromeric protein B) in BRCA1-deficient UWB1.289 cells containing alphoid tetO -HAC /BRCA1 and in the same cells that have lost the HAC. ( f ) Quantitative RT-PCR experiments showing the transcripts of the double homeobox 4 ( DUX4 ) gene located in the heterochromatic subtelomeric 4q35 region, the cancer-testis SPANX-B gene located at the Xq27 region and, as a control, a housekeeping gene PPIH, peptidyprolyl isomerase H (cyclophilin H) was used. Asterisk denotes significant difference.
    Figure Legend Snippet: De-repression of alpha-satellite DNA transcription in BRCA1-deficient UWB1.289 cells. ( a , c and d ) Quantitative RT-PCR experiments showing that the satellite DNA transcripts of HORs regions of the chromosome 21 (chr21a and chr21b), chromosome X (chrX) ( a ), chromosome 5 (D5Z1 and D5Z2) ( d ) and DNA transcripts of pericentromeric Sat2 repeats located at 10q21 ( c ) are significantly repressed in the cells carrying alphoid tetO -HAC/BRCA1 compared to that of the cells that have lost the HAC. C t values were normalized relative to each other. Error bars indicate SD. ( b ) Timing of transcription of HORs regions of chromosome X and chromosome 21. ( e ) ChIP analysis of different centromeric regions using antibodies against CENP-B (centromeric protein B) in BRCA1-deficient UWB1.289 cells containing alphoid tetO -HAC /BRCA1 and in the same cells that have lost the HAC. ( f ) Quantitative RT-PCR experiments showing the transcripts of the double homeobox 4 ( DUX4 ) gene located in the heterochromatic subtelomeric 4q35 region, the cancer-testis SPANX-B gene located at the Xq27 region and, as a control, a housekeeping gene PPIH, peptidyprolyl isomerase H (cyclophilin H) was used. Asterisk denotes significant difference.

    Techniques Used: Quantitative RT-PCR, HAC Assay, Chromatin Immunoprecipitation

    Expression of the human BRCA1 gene in porcine ST cells. ( a ) From hamster CHO cells exhibiting a high efficiency of microcell formation, the alphoid tetO -HAC/BRCA1 was transferred into the pig's testis fibroblast ST cells using MMCT technique. (b) FISH analysis of porcine cells containing the alphoid tetO -HAC/BRCA1. Chromosomal DNA was counterstained with DAPI (blue). Arrow indicates to the HAC. ( c ) Fluorescence image of cells carrying the alphoid tetO -HAC/BRCA1 containing the expressed eGFP transgene. ( d ) Expression of the human BRCA1 gene in porcine cells. RT-PCR analysis of genomic DNA isolated from the original ST cells (lane 4), from two porcine clones (pl4 and pl5) containing the alphoid tetO -HAC/BRCA1 (lanes 2 and 3) and from the human HeLa cells as a positive control (lane 1) using the human BRCA1-specific primers (Supplementary Table S1). The size of the predicted product is 839 bp. M-ladder containing alphoid tetO -HAC/BRCA1
    Figure Legend Snippet: Expression of the human BRCA1 gene in porcine ST cells. ( a ) From hamster CHO cells exhibiting a high efficiency of microcell formation, the alphoid tetO -HAC/BRCA1 was transferred into the pig's testis fibroblast ST cells using MMCT technique. (b) FISH analysis of porcine cells containing the alphoid tetO -HAC/BRCA1. Chromosomal DNA was counterstained with DAPI (blue). Arrow indicates to the HAC. ( c ) Fluorescence image of cells carrying the alphoid tetO -HAC/BRCA1 containing the expressed eGFP transgene. ( d ) Expression of the human BRCA1 gene in porcine cells. RT-PCR analysis of genomic DNA isolated from the original ST cells (lane 4), from two porcine clones (pl4 and pl5) containing the alphoid tetO -HAC/BRCA1 (lanes 2 and 3) and from the human HeLa cells as a positive control (lane 1) using the human BRCA1-specific primers (Supplementary Table S1). The size of the predicted product is 839 bp. M-ladder containing alphoid tetO -HAC/BRCA1

    Techniques Used: Expressing, HAC Assay, Fluorescence In Situ Hybridization, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Isolation, Clone Assay, Positive Control

    30) Product Images from "An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation"

    Article Title: An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0407-4

    Viral integration levels determined in the presence and absence of NSC and integration inhibitor. Jurkat cells were pre-treated with NSC [100 µM] or raltegravir [1 µM], and infected with LAI 6 h post-treatment in the presence or absence of 50 µM NSC or 500 nM raltegravir. 24 h post-infection, cells were harvested and subjected to DNA extraction and HIV-1 integration was assayed by Alu - gag PCR followed by quantification of integration events by HIV-1 gag qPCR. The number of integration events relative to LAI-infected cells (WT) in the presence of NSC or raltegravir is shown. For each sample, variations in the amount and quality of cellular DNA input was accounted for by dividing the number of HIV-1 integration events detected by the HIV-1 gag qPCR by the relative actin level detected in the input DNA for the Alu - gag PCR. All samples were then normalized relative to one another such that the average WT value was 1. The average of three samples is shown for each condition. Error bars represent SD. *Statistically significant from WT by Student’s t test, p
    Figure Legend Snippet: Viral integration levels determined in the presence and absence of NSC and integration inhibitor. Jurkat cells were pre-treated with NSC [100 µM] or raltegravir [1 µM], and infected with LAI 6 h post-treatment in the presence or absence of 50 µM NSC or 500 nM raltegravir. 24 h post-infection, cells were harvested and subjected to DNA extraction and HIV-1 integration was assayed by Alu - gag PCR followed by quantification of integration events by HIV-1 gag qPCR. The number of integration events relative to LAI-infected cells (WT) in the presence of NSC or raltegravir is shown. For each sample, variations in the amount and quality of cellular DNA input was accounted for by dividing the number of HIV-1 integration events detected by the HIV-1 gag qPCR by the relative actin level detected in the input DNA for the Alu - gag PCR. All samples were then normalized relative to one another such that the average WT value was 1. The average of three samples is shown for each condition. Error bars represent SD. *Statistically significant from WT by Student’s t test, p

    Techniques Used: Infection, DNA Extraction, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    31) Product Images from "Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae"

    Article Title: Binding site number variation and high-affinity binding consensus of Myb-SANT-like transcription factor Adf-1 in Drosophilidae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq504

    In vivo binding of ADF-1 at the D. funebris Adh promoter region. ( A ) Quantification of ADF-1 binding sites. The input sample is indicated in white, the anti-ADF-1 ChIP in gray and the control ChIP in black. ( B ) Enrichment of ADF-1 binding sites in anti-ADF-1 ChIP relative to the control IgG ChIP experiments. ( C ) The total fraction of specifically immunoprecipitated DNA with respect to the input sample. A-IP, anti-ADF-1 ChIP; I-IP, control ChIP experiment with unspecific IgG.
    Figure Legend Snippet: In vivo binding of ADF-1 at the D. funebris Adh promoter region. ( A ) Quantification of ADF-1 binding sites. The input sample is indicated in white, the anti-ADF-1 ChIP in gray and the control ChIP in black. ( B ) Enrichment of ADF-1 binding sites in anti-ADF-1 ChIP relative to the control IgG ChIP experiments. ( C ) The total fraction of specifically immunoprecipitated DNA with respect to the input sample. A-IP, anti-ADF-1 ChIP; I-IP, control ChIP experiment with unspecific IgG.

    Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation

    32) Product Images from "The ATM and Rad3-Related (ATR) Protein Kinase Pathway Is Activated by Herpes Simplex Virus 1 and Required for Efficient Viral Replication"

    Article Title: The ATM and Rad3-Related (ATR) Protein Kinase Pathway Is Activated by Herpes Simplex Virus 1 and Required for Efficient Viral Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.01884-17

    Inhibition of either ATR or Chk1 kinase activity significantly inhibits HSV-1 replication and virus production. (A) Quantification of HSV-1 PFU following inhibitor treatment shows that all ATR and Chk1 inhibitors tested, and the control polymerase inhibitor aphidicolin, significantly impact virus production. Inhibitors of ATM, Chk2, or DNA-PK were not found to significantly inhibit virus production. Asterisks indicate statistical significance (Student's t test): P ≤ 0.005 in all cases except for PF477736, for which the P value was 0.006. (B) qPCR measurements of HSV-1 genome copies show good agreement with the viral titer assays.
    Figure Legend Snippet: Inhibition of either ATR or Chk1 kinase activity significantly inhibits HSV-1 replication and virus production. (A) Quantification of HSV-1 PFU following inhibitor treatment shows that all ATR and Chk1 inhibitors tested, and the control polymerase inhibitor aphidicolin, significantly impact virus production. Inhibitors of ATM, Chk2, or DNA-PK were not found to significantly inhibit virus production. Asterisks indicate statistical significance (Student's t test): P ≤ 0.005 in all cases except for PF477736, for which the P value was 0.006. (B) qPCR measurements of HSV-1 genome copies show good agreement with the viral titer assays.

    Techniques Used: Inhibition, Activity Assay, Real-time Polymerase Chain Reaction

    33) Product Images from "High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies"

    Article Title: High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025181

    ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.
    Figure Legend Snippet: ChIP-chip results obtained from mouse embryonic brain. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Example of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. Contrarily to N2a cells, in which several continuous probes were often found enriched, only one or two probes were found enriched per gene. C) Venn diagram illustrating the overlap (black) between Arx-immunoprecipitated genes in transfected N2a cells (blue) and mouse embryonic brain (red), and the number of genes with at least 75% match to Arx-binding motif.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Binding Assay

    ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).
    Figure Legend Snippet: ChIP-chip results obtained from Arx-transfected N2a cells. A) Graph representing log 2 probe intensities of Arx-immunoprecipitated DNA (IP) and input DNA obtained in a representative ChIP experiment. The red dots indicate the probes enriched in Arx-immunoprecipitates compared to total input DNA. B) Examples of the enrichment profiles of Arx-bound promoter regions visualized by DNA analytics software. The 3 lines represent data obtained from 3 independent ChIP-chip experiments and show the reproducibility between the 3 replicates. C) The resulting weighted matrix discovered through the MDModule analysis (top) appears to be similar to the motif identified by Berger et al. [27] (bottom). D) Frequency distribution of scores. The TAATTA motif identified by MDModule was significantly more present in ChIP-enriched sequences (red curve) by comparison to negative control sequences (blue curve).

    Techniques Used: Chromatin Immunoprecipitation, Transfection, Immunoprecipitation, Software, Negative Control

    Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.
    Figure Legend Snippet: Confirmation of Arx binding to candidate promoter regions. A) Arx-immunoprecipitated DNA was compared to input DNA by ChIP/QFM-PCR to determine ChIP enrichment of 21 putative target genes. No enrichment occurred for the Vapb promoter which was consistently negative in all ChIP experiments. Bar heights represent log 10 enrichment of the signal obtained for Arx-immunoprecipitated DNA versus input DNA for each promoter using site-specific primers. The arrows above the bars indicate sequences in which the previously defined Arx-binding motif was found. B) Confirmation of Arx binding to 5 different promoter sequences identified by ChIP using a luciferase reporter gene assay. Firefly luciferase data were normalized to Renilla luciferase expression and data are presented as the percentage of transcriptional activity compared to the vector control. Arx regulation was confirmed for Lmo1 , Lmo3 , Sh3tc2 , Calb2 and Cdh2 promoter regions as transcriptional activity was repressed in the presence of Arx by comparison to the control transfection, whereas it had no effect on the plasmid alone pGL4.23. Error bars indicate SEM.

    Techniques Used: Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Reporter Gene Assay, Expressing, Activity Assay, Plasmid Preparation, Transfection

    34) Product Images from "Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts"

    Article Title: Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-015-0151-8

    TGFβ1 increased H3K4me1/3 levels on TGFBIp and ECM-associated gene promoters in wild-type and GCD2-homozygous cells. a , b Bar graphs showing H3K4me3 ( a ) and H3K4me1 ( b ) levels at the indicated gene promoters in control and TGFβ1 (5 ng/ml)-stimulated wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K4me1 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are shown as the mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: TGFβ1 increased H3K4me1/3 levels on TGFBIp and ECM-associated gene promoters in wild-type and GCD2-homozygous cells. a , b Bar graphs showing H3K4me3 ( a ) and H3K4me1 ( b ) levels at the indicated gene promoters in control and TGFβ1 (5 ng/ml)-stimulated wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K4me1 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are shown as the mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    MLL1 knockdown attenuated TGFβ1-induced increases in H3K4me3 on the promoters of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGF-β1 (5 ng/ml) for 8 h, and H3K4me3 levels at the indicated gene promoters were analyzed. ChIP assays were performed with H3K4me3 antibody. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are presented as mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: MLL1 knockdown attenuated TGFβ1-induced increases in H3K4me3 on the promoters of TGFBIp and ECM-associated genes. a Wild-type and GCD2-homozygous corneal fibroblasts were infected with MLL1 or control shRNA lentivirus. After puromycin selection, infected cells were stimulated with TGF-β1 (5 ng/ml) for 8 h, and H3K4me3 levels at the indicated gene promoters were analyzed. ChIP assays were performed with H3K4me3 antibody. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the indicated gene promoters to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are presented as mean fold change of the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Infection, shRNA, Selection, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    TGFBIp transcription and H3K4me3 levels in wild-type and GCD2-homozygous corneal fibroblasts. a , b Slit-lamp photographs and pedigree of wild-type, GCD2 heterozygous, and GCD2 homozygous cells. c , d The mRNA and protein levels of TGFBIp in normal and GCD2 corneal fibroblasts was determined by RT-qPCR ( c ) and western blot ( d ). Gene expression was normalized to internal control GAPDH gene, and results are expressed as fold stimulation over control. e , f H3K4me3 and H3K27me3 levels at the Smad binding elements on TGFBIp gene promoters and at the TSS of TGFBIp gene in wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K27me3 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the TGFBIp gene promoter to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are the mean fold change relative to the input ± standard error (SE) of enrichment. (mean ± SE; ** P
    Figure Legend Snippet: TGFBIp transcription and H3K4me3 levels in wild-type and GCD2-homozygous corneal fibroblasts. a , b Slit-lamp photographs and pedigree of wild-type, GCD2 heterozygous, and GCD2 homozygous cells. c , d The mRNA and protein levels of TGFBIp in normal and GCD2 corneal fibroblasts was determined by RT-qPCR ( c ) and western blot ( d ). Gene expression was normalized to internal control GAPDH gene, and results are expressed as fold stimulation over control. e , f H3K4me3 and H3K27me3 levels at the Smad binding elements on TGFBIp gene promoters and at the TSS of TGFBIp gene in wild-type and GCD2-homozygous corneal fibroblasts. ChIP assays were performed with H3K4me3 and H3K27me3 antibodies. Immunoprecipitated DNA and input DNA were subjected to qPCR with primers specific for the TGFBIp gene promoter to measure enrichment levels. qPCR data were analyzed using the 2 -ΔΔCt method, and data are the mean fold change relative to the input ± standard error (SE) of enrichment. (mean ± SE; ** P

    Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    35) Product Images from "Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue"

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

    Journal: Biological psychiatry

    doi: 10.1016/j.biopsych.2016.03.1048

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score
    Figure Legend Snippet: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Fluorescence, FACS, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Ligation

    36) Product Images from "Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: Possible reaction of cadmium with the zinc finger domain of Sp1"

    Article Title: Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: Possible reaction of cadmium with the zinc finger domain of Sp1

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2009.12.038

    Exposure of PMKC to Cd reduces in vivo binding of Sp1 to the GC boxes in the promoters of SGLT1 and SGLT2 genes PMKC were either left untreated (M) or treated for 24 hr with 5 μM CdCl 2 (Cd). ChIP assay was performed using Sp1 antibody (12 μg) (IP) or with un-immunized rabbit serum for SGLT1 and without antibody for SGLT2 as background. PCR was performed on input DNA and on chromatin DNA isolated by ChIP using primers specific to either the GC-1 site of SGLT1 ( A ) or to the GC site of SGLT2 ( C ) genes. The intensity of the PCR bands from ChIP assays were normalized to the input DNA, and then background values were subtracted from that of the chromatin immunoprecipitated with Sp1 and graphed ( B,D ). L, 20 bp DNA ladder. ( N = 5 for SGLT1 ; N = 3 for SGLT2 ).
    Figure Legend Snippet: Exposure of PMKC to Cd reduces in vivo binding of Sp1 to the GC boxes in the promoters of SGLT1 and SGLT2 genes PMKC were either left untreated (M) or treated for 24 hr with 5 μM CdCl 2 (Cd). ChIP assay was performed using Sp1 antibody (12 μg) (IP) or with un-immunized rabbit serum for SGLT1 and without antibody for SGLT2 as background. PCR was performed on input DNA and on chromatin DNA isolated by ChIP using primers specific to either the GC-1 site of SGLT1 ( A ) or to the GC site of SGLT2 ( C ) genes. The intensity of the PCR bands from ChIP assays were normalized to the input DNA, and then background values were subtracted from that of the chromatin immunoprecipitated with Sp1 and graphed ( B,D ). L, 20 bp DNA ladder. ( N = 5 for SGLT1 ; N = 3 for SGLT2 ).

    Techniques Used: In Vivo, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Isolation, Immunoprecipitation

    37) Product Images from "False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures"

    Article Title: False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2014.06.001

    Next-generation sequencing mispriming events correlate with low input DNA and low read depth. Modified box plots of input DNA ( A ) and read depth ( B ) versus percentage of misprimed reads at EGFR exon 21 (G873R). The horizontal line in the middle of each
    Figure Legend Snippet: Next-generation sequencing mispriming events correlate with low input DNA and low read depth. Modified box plots of input DNA ( A ) and read depth ( B ) versus percentage of misprimed reads at EGFR exon 21 (G873R). The horizontal line in the middle of each

    Techniques Used: Next-Generation Sequencing, Modification

    38) Product Images from "A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses"

    Article Title: A new plant-specific syntaxin-6 protein may define an intracytoplasmic route for begomoviruses

    Journal: bioRxiv

    doi: 10.1101/2020.01.10.901496

    NISP interacts with NSP in vivo. (A) NISP interacts with NSP in vesicle-like compartments, as judged by BiFC. N. benthamiana leaf cells expressing NISP and NSP fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP, or in combination with the empty vectors, were observed by confocal microscopy 3 days after infiltration. Scale bars, 10 μm. The experiment was repeated at least four times with identical results. (B) NISP-NSP complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NISP- and cYFP-NSP-expressing DNA constructs. Images were taken 2 days post agroinfiltration and 30 min after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64. Scale bars, 10 μm. The experiment was repeated three times with similar results. (C) NISP interacts with NSP in planta . Whole cell protein extracts from N. benthamiana leaves expressing transiently NISP-GFP and NSP-6HA were used for co-immunoprecipitation assays using an anti-GFP serum. Input and IP show the levels of the expressed proteins NSP-6HA, NISP-GFP, and GFP. Anti-HA antibody was used to detect NSP-6HA from the immunoprecipitated complex. GFP was used as a negative control. Molecular mass markers are given on the left in kilodaltons. The experiment was repeated twice. (D) In vivo interaction of NISP with NSP. Co-immunoprecipitation was performed as described in B, except that the tags of the transiently expressed proteins were switched to NSP-GFP and NISP-6HA. The experiment was repeated twice.
    Figure Legend Snippet: NISP interacts with NSP in vivo. (A) NISP interacts with NSP in vesicle-like compartments, as judged by BiFC. N. benthamiana leaf cells expressing NISP and NSP fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP, or in combination with the empty vectors, were observed by confocal microscopy 3 days after infiltration. Scale bars, 10 μm. The experiment was repeated at least four times with identical results. (B) NISP-NSP complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NISP- and cYFP-NSP-expressing DNA constructs. Images were taken 2 days post agroinfiltration and 30 min after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64. Scale bars, 10 μm. The experiment was repeated three times with similar results. (C) NISP interacts with NSP in planta . Whole cell protein extracts from N. benthamiana leaves expressing transiently NISP-GFP and NSP-6HA were used for co-immunoprecipitation assays using an anti-GFP serum. Input and IP show the levels of the expressed proteins NSP-6HA, NISP-GFP, and GFP. Anti-HA antibody was used to detect NSP-6HA from the immunoprecipitated complex. GFP was used as a negative control. Molecular mass markers are given on the left in kilodaltons. The experiment was repeated twice. (D) In vivo interaction of NISP with NSP. Co-immunoprecipitation was performed as described in B, except that the tags of the transiently expressed proteins were switched to NSP-GFP and NISP-6HA. The experiment was repeated twice.

    Techniques Used: In Vivo, Bimolecular Fluorescence Complementation Assay, Expressing, Confocal Microscopy, Staining, Construct, Immunoprecipitation, Negative Control

    NISP displays a pro-begomoviral function. (A) Transcript accumulation of NISP in transgenic lines. NISP transcript levels in three independently complemented transgenic lines ( nisp-1 /NISP-1-3), three independently transformed NISP -overexpressing lines (AtNISP1-3), Col-0 and nisp-1 were determined by qRT-PCR. Gene expression was calculated using the 2 -ΔCt method, and actin was used as an endogenous control. Error bars represent 95% confidence intervals based on replicated samples (n=3) from three independent experiments. (B) NISP-GFP protein accumulation in transgenic lines. Whole cell protein extracts from the complemented transgenic lines ( nisp-1 /NISP), NISP -overexpressing lines (AtNISP), Col-0 and nisp-1 were separated by SDS-PAGE and immunoblotted using an anti-GFP serum. (C) AT2G18860 transcript accumulation in transgenic lines. The transcript levels of AT2G18860 in complemented transgenic lines ( at2g18860-1 /AT2G18860-1-3), AT2G18860-overexpressing lines (AT2G18860-1-3), Col-0 and at2g18860-1 were quantified by qRT-PCR and normalized to the actin as an endogenous control. Error bars indicate 95% confidence intervals based on replicated samples (n=3) from three independent experiments. (D) AT2G18860-GFP protein accumulation in transgenic lines. Total protein extracts from complemented transgenic lines ( at2g18860-1 /AT2G18860), AT2G18860-overexpressing lines (AT2G18860), Col-0 and at2g18860-1 were fractionated by SDS-PAGE and immunoblotted using anti-GFP antibody. (E) CaLCuV infection-associated symptoms in Arabidopsis genotypes at 21 days post-inoculation (dpi). The figure shows representative samples of mock-inoculated and CaLCuV -infected plants. The genotypes are indicated in the figure and are the same as specified in A-D. (F) The onset of infection is delayed in nisp-1 knockout line. Ecotype Col-0, nisp-1 , at2g18860-1 , nisp-1 /NISP complemented lines and AtNISP-overexpressing lines (as in a-d) were infected with CaLCuV DNA, and the course of infection was monitored as the percent of systemically infected plants at different dpi. (G) Absolute quantification of CaLCuV genomic units in infected lines at 21 dpi. The genotypes are the same as specified in a-d. Error bars indicate 95% confidence intervals based on replicated samples from three independent experiments. Different letters indicate significative differences among the groups (Duncan test, p
    Figure Legend Snippet: NISP displays a pro-begomoviral function. (A) Transcript accumulation of NISP in transgenic lines. NISP transcript levels in three independently complemented transgenic lines ( nisp-1 /NISP-1-3), three independently transformed NISP -overexpressing lines (AtNISP1-3), Col-0 and nisp-1 were determined by qRT-PCR. Gene expression was calculated using the 2 -ΔCt method, and actin was used as an endogenous control. Error bars represent 95% confidence intervals based on replicated samples (n=3) from three independent experiments. (B) NISP-GFP protein accumulation in transgenic lines. Whole cell protein extracts from the complemented transgenic lines ( nisp-1 /NISP), NISP -overexpressing lines (AtNISP), Col-0 and nisp-1 were separated by SDS-PAGE and immunoblotted using an anti-GFP serum. (C) AT2G18860 transcript accumulation in transgenic lines. The transcript levels of AT2G18860 in complemented transgenic lines ( at2g18860-1 /AT2G18860-1-3), AT2G18860-overexpressing lines (AT2G18860-1-3), Col-0 and at2g18860-1 were quantified by qRT-PCR and normalized to the actin as an endogenous control. Error bars indicate 95% confidence intervals based on replicated samples (n=3) from three independent experiments. (D) AT2G18860-GFP protein accumulation in transgenic lines. Total protein extracts from complemented transgenic lines ( at2g18860-1 /AT2G18860), AT2G18860-overexpressing lines (AT2G18860), Col-0 and at2g18860-1 were fractionated by SDS-PAGE and immunoblotted using anti-GFP antibody. (E) CaLCuV infection-associated symptoms in Arabidopsis genotypes at 21 days post-inoculation (dpi). The figure shows representative samples of mock-inoculated and CaLCuV -infected plants. The genotypes are indicated in the figure and are the same as specified in A-D. (F) The onset of infection is delayed in nisp-1 knockout line. Ecotype Col-0, nisp-1 , at2g18860-1 , nisp-1 /NISP complemented lines and AtNISP-overexpressing lines (as in a-d) were infected with CaLCuV DNA, and the course of infection was monitored as the percent of systemically infected plants at different dpi. (G) Absolute quantification of CaLCuV genomic units in infected lines at 21 dpi. The genotypes are the same as specified in a-d. Error bars indicate 95% confidence intervals based on replicated samples from three independent experiments. Different letters indicate significative differences among the groups (Duncan test, p

    Techniques Used: Transgenic Assay, Transformation Assay, Quantitative RT-PCR, Expressing, SDS Page, Infection, Knock-Out

    NISP complexes with vDNA in vivo . (A) NISP-GFP accumulation in overexpressing lines. Whole cell protein extracts from the NISP-GFP-overexpressing lines, as indicated in the figure, and Col-0 were separated by SDS-PAGE and immunoblotted using an anti-GFP serum. (B) NISP binds to viral DNA-B in vivo . NISP-GFP-overexpressing lines (AtNISP-1, AtNISP-2, and AtNISP-3), and Col-0 were inoculated with infectious CaLCuV clones prior to the ChIP assay, which was performed with leaf samples of the indicated genotypes using anti-GFP. The infected plants were confirmed by PCR of input DNA from inoculated plants using CaLCuV DNA-B-specific primers, which amplifies a 770-bp fragment (F1/R1; S10b Fig). IN denotes infected leaves and UN, uninfected leaves. (C) ChIP-qPCR assay of leaves from AtNISP-GFP transgenic lines and Col-0. The samples were immunoprecipitated with an anti-GFP antibody and ChIPed DNA was quantified by qPCR using the DNA-B-specific primers F3/R3 (S10b Fig). Data were normalized relative to the input of each sample and are expressed as the percentage of input. (D) ChIP-qPCR assay using a different set of DNA-B-specific primers (F2/R2) and samples from the indicated genotypes. The ChIP-qPCR was performed as described in C. (E) NISP binds to viral DNA-A in vivo . NISP-GFP-overexpressing lines (AtNISP-1, AtNISP-2 and AtNISP-3), and Col-0 were inoculated with infectious CaLCuV clones prior to the ChIP assay, which was performed with leaf samples of the indicated genotypes using anti-GFP. The infected plants were confirmed by PCR of input DNA from inoculated plants using CaLCuV DNA-A-specific primers, which amplifies a 389-bp fragment (F1/R1; S10a Fig). IN denotes infected leaves and UN, uninfected leaves. (F) ChIP-qPCR assay using a set of DNA-A-specific primers (F2/R2, S10a Fig) and samples from the indicated genotypes. The ChIP-qPCR was performed as described in C. The above experiments were repeated three times with similar results.
    Figure Legend Snippet: NISP complexes with vDNA in vivo . (A) NISP-GFP accumulation in overexpressing lines. Whole cell protein extracts from the NISP-GFP-overexpressing lines, as indicated in the figure, and Col-0 were separated by SDS-PAGE and immunoblotted using an anti-GFP serum. (B) NISP binds to viral DNA-B in vivo . NISP-GFP-overexpressing lines (AtNISP-1, AtNISP-2, and AtNISP-3), and Col-0 were inoculated with infectious CaLCuV clones prior to the ChIP assay, which was performed with leaf samples of the indicated genotypes using anti-GFP. The infected plants were confirmed by PCR of input DNA from inoculated plants using CaLCuV DNA-B-specific primers, which amplifies a 770-bp fragment (F1/R1; S10b Fig). IN denotes infected leaves and UN, uninfected leaves. (C) ChIP-qPCR assay of leaves from AtNISP-GFP transgenic lines and Col-0. The samples were immunoprecipitated with an anti-GFP antibody and ChIPed DNA was quantified by qPCR using the DNA-B-specific primers F3/R3 (S10b Fig). Data were normalized relative to the input of each sample and are expressed as the percentage of input. (D) ChIP-qPCR assay using a different set of DNA-B-specific primers (F2/R2) and samples from the indicated genotypes. The ChIP-qPCR was performed as described in C. (E) NISP binds to viral DNA-A in vivo . NISP-GFP-overexpressing lines (AtNISP-1, AtNISP-2 and AtNISP-3), and Col-0 were inoculated with infectious CaLCuV clones prior to the ChIP assay, which was performed with leaf samples of the indicated genotypes using anti-GFP. The infected plants were confirmed by PCR of input DNA from inoculated plants using CaLCuV DNA-A-specific primers, which amplifies a 389-bp fragment (F1/R1; S10a Fig). IN denotes infected leaves and UN, uninfected leaves. (F) ChIP-qPCR assay using a set of DNA-A-specific primers (F2/R2, S10a Fig) and samples from the indicated genotypes. The ChIP-qPCR was performed as described in C. The above experiments were repeated three times with similar results.

    Techniques Used: In Vivo, SDS Page, Clone Assay, Chromatin Immunoprecipitation, Infection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transgenic Assay, Immunoprecipitation

    NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.
    Figure Legend Snippet: NISP interacts with NIG in vivo and NSP enhances NISP-NIG complex formation. (A) BIFC assay showing the interaction between NISP and NIG in vesicles of N. benthamiana leaf cells. N. benthamiana leaves transiently expressing NISP and NIG fused to the C-terminus (cYFP) or N-terminus (nYFP) of YFP were examined by confocal microscopy 3 days after infiltration. Scale bars, 10 μm and 20 μm. The experiment was repeated three times with similar results. (B) NISP-NIG complex-containing vesicles are stained by FM4-64. N. benthamiana leaves were agroinfiltrated with nYFP-NIG- and cYFP-NISP-expressing DNA constructs. Images were taken 2 days post infiltration and 1 h after infiltration of FM4-64. Arrows indicate examples of vesicle-associated reconstituted fluorescent signals that were co-stained by FM4-64 and the asterisk, the region of the amplified inset. Scale bars, 10 μm. The experiment was repeated three times with identical results. (C) In vivo interaction of NISP and NIG. Total protein extracts from N. benthamiana expressing NISP-GFP and NIG-6HA were used for co-immunoprecipitation assays using anti-GFP. Input and IP show the levels of the expressed proteins NISP-GFP and NIG-6HA. Anti-HA was used to detect NIG-6HA from the immunoprecipitated complex. GFP was used as an unrelated protein. The experiment was repeated twice with identical results. (D) NISP-NIG complex formation in the presence of viral NSP. The Co-IP assay was performed as described in A, except that co-expressed NSP-GST was included in the assay. NSP-GST was detected by immunoprecipitating it from infiltrated leaves and immunoblotting with anti-GST. The experiment was repeated twice with identical results. (E) Viral NSP enhances the interaction between NISP and NIG. NIG levels in the immunoprecipitated complex in the presence and absence of viral NSP were quantified using the Band Analysis tools of the ImageLab software (Bio-Rad). The signal values were normalized using the IP NISP-GFP band. A.U. denotes arbitrary units.

    Techniques Used: In Vivo, Bimolecular Fluorescence Complementation Assay, Expressing, Confocal Microscopy, Staining, Construct, Amplification, Immunoprecipitation, Co-Immunoprecipitation Assay, Software

    39) Product Images from "Possible role of human herpesvirus 8 in the lymphoproliferative disorders in common variable immunodeficiency"

    Article Title: Possible role of human herpesvirus 8 in the lymphoproliferative disorders in common variable immunodeficiency

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20050381

    Detection of HHV8 genomes by nested PCR from PBMCs. Dotted lines separate patient/subject groups. The cohorts of patients included: “Normal” (normal blood donors), “IVIG” (patients receiving intravenous immunoglobulin for disorders other than CVID), “CVID-control” (CVID patients without GLILD), and “CVID-GLILD” (CVID patients with GLILD). + (lane 12) is BCBL-1, an HHV8-infected B cell lymphoma cell line (positive control; lane1) is an H 2 O template negative control. ORF26 and ORFK9 indicate HHV8 open reading frames 26 and K9, respectively. β-actin indicates PCR of patient DNA using primers specific for β-actin. HHV8 amplicons were detected from DNA by nested PCR (PBMC DNA) or nonnested PCR (BCBL-1 DNA [lane 12]). White lines indicate that intervening lanes have been spliced out.
    Figure Legend Snippet: Detection of HHV8 genomes by nested PCR from PBMCs. Dotted lines separate patient/subject groups. The cohorts of patients included: “Normal” (normal blood donors), “IVIG” (patients receiving intravenous immunoglobulin for disorders other than CVID), “CVID-control” (CVID patients without GLILD), and “CVID-GLILD” (CVID patients with GLILD). + (lane 12) is BCBL-1, an HHV8-infected B cell lymphoma cell line (positive control; lane1) is an H 2 O template negative control. ORF26 and ORFK9 indicate HHV8 open reading frames 26 and K9, respectively. β-actin indicates PCR of patient DNA using primers specific for β-actin. HHV8 amplicons were detected from DNA by nested PCR (PBMC DNA) or nonnested PCR (BCBL-1 DNA [lane 12]). White lines indicate that intervening lanes have been spliced out.

    Techniques Used: Nested PCR, Infection, Positive Control, Negative Control, Polymerase Chain Reaction

    40) Product Images from "The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma"

    Article Title: The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma

    Journal: Scientific Reports

    doi: 10.1038/srep22113

    SS18-SSX promotes Histone H3K9 acetylation and chromatin accessibility at the AXIN2 promoter. ( A ) Histone H3K9 Ac ChIP in C3H10T1/2 pLIVc (pLIVc), C3H10T1/ 2 SS18-SSX1-V5 (SS-V5) and STO cells infected with SS18-SSX-V5 or empty pLIVc. Cross-linked chromatin was sonicated and immunoprecipitated with an anti Histone H3K9 Ac antibody or rabbit IgG. Co-immunoprecipitated DNA was quantified by real-time PCR using primer pairs annealing to the mouse AXIN2 promoter region at the indicated positions. Results are expressed as fold enrichment of values obtained with rabbit IgG precipitates after normalization for the total amount of input chromatin. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests. ( B ) Chromatin accessibility tests in C3H10T1/2 pLIVc (pLIVc), C3H10T1/ 2 SS18-SSX1-V5 (SS-V5) and C3H10T1/ 2 SS18-SS1-HA (SS-HA) cells. Positions of primers used for amplification of MspI CHART products by qRT-PCR are reported with approximate location of ATGs and transcription factor binding sites. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests.
    Figure Legend Snippet: SS18-SSX promotes Histone H3K9 acetylation and chromatin accessibility at the AXIN2 promoter. ( A ) Histone H3K9 Ac ChIP in C3H10T1/2 pLIVc (pLIVc), C3H10T1/ 2 SS18-SSX1-V5 (SS-V5) and STO cells infected with SS18-SSX-V5 or empty pLIVc. Cross-linked chromatin was sonicated and immunoprecipitated with an anti Histone H3K9 Ac antibody or rabbit IgG. Co-immunoprecipitated DNA was quantified by real-time PCR using primer pairs annealing to the mouse AXIN2 promoter region at the indicated positions. Results are expressed as fold enrichment of values obtained with rabbit IgG precipitates after normalization for the total amount of input chromatin. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests. ( B ) Chromatin accessibility tests in C3H10T1/2 pLIVc (pLIVc), C3H10T1/ 2 SS18-SSX1-V5 (SS-V5) and C3H10T1/ 2 SS18-SS1-HA (SS-HA) cells. Positions of primers used for amplification of MspI CHART products by qRT-PCR are reported with approximate location of ATGs and transcription factor binding sites. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests.

    Techniques Used: Chromatin Immunoprecipitation, Infection, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Binding Assay

    DNA dependence of SS18-SSX interactions. ( A ) Graphical representation and statistical analysis of PLAs using antibodies against the indicated proteins. C3H10T1/ 2 SS18-SSX1-V5 cells were pre-stained with 5 μM DAPI for 30 min and subjected to PLA or subjected to PLA and then counterstained with DAPI. PLA signal quantification was performed as described in materials and methods. ( B ) ChIP using anti-TCF3/4, anti-V5 and anti-HDAC1 antibodies in C3H10T1/2 pLIVc and C3H10T1/ 2 SS18-SSX1-V5 cells. Results are expressed as fold enrichment of values obtained with rabbit or mouse IgG immunoprecipitates, after normalization for the total amount of input chromatin. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests.
    Figure Legend Snippet: DNA dependence of SS18-SSX interactions. ( A ) Graphical representation and statistical analysis of PLAs using antibodies against the indicated proteins. C3H10T1/ 2 SS18-SSX1-V5 cells were pre-stained with 5 μM DAPI for 30 min and subjected to PLA or subjected to PLA and then counterstained with DAPI. PLA signal quantification was performed as described in materials and methods. ( B ) ChIP using anti-TCF3/4, anti-V5 and anti-HDAC1 antibodies in C3H10T1/2 pLIVc and C3H10T1/ 2 SS18-SSX1-V5 cells. Results are expressed as fold enrichment of values obtained with rabbit or mouse IgG immunoprecipitates, after normalization for the total amount of input chromatin. Results are representative of three independent experiments. Error bars represent the SD of triplicate PCR tests.

    Techniques Used: Staining, Proximity Ligation Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

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    Article Snippet: .. Quantitative real-time PCR was performed on input DNA and antibody-specific ChIP DNA using SYBR Green Master Mix reagents with an ABI Step one plus thermocycler (Applied Biosystems, Foster City, CA) detection system. .. Enrichment of RB family members at target gene promoters was examined using primers spanning known E2F binding sites at the murine CCNA2 and MCM10 loci.

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: .. Purified DNA from immunoprecipitates and from input DNA was analyzed by quantitative real-time PCR on an Applied Biosystems 7500 Real-Time PCR System using Power SYBR® Green PCR Master Mix (Applied Biosystems). ..

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue
    Article Snippet: .. These tests include 1) Agilent Bioanalyzer of the input DNA ( ); 2) Qubit fluorometric assay (Thermo Fisher Scientific) concentration measurement of the input DNA and ChIP DNA product; and 3) qPCR of the input DNA and ChIP DNA product ( ). .. First, successful chromatin digestion, resulting in an overwhelming share of mononucleosomes, should be confirmed using Agilent Bioanalyzer (the trace will show the major DNA fragment around 150 bp) ( ).

    Article Title: BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation
    Article Snippet: .. Both immunoprecipitated DNA and input DNA were analyzed by real-time quantitative PCR (qPCR; Applied Biosystems). .. Monoclonal anti-myc antibody (Sigma-Aldrich) was used.

    Transfection:

    Article Title: A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication
    Article Snippet: .. To eliminate the transfected input DNA, the RNA preparation was further treated with 2 U RNase-free recombinant DNase I for 30 minutes at 37°C by using the DNA-free Kit (Ambion), followed by re-suspension in RNase-free H2 O. RT-PCR was employed to detect the (-) gRNAs and sg mRNAs using specific primers. ..

    Amplification:

    Article Title: BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer
    Article Snippet: .. PCR reactions for amplification of chromatin immunoprecipitates and input DNA were performed in a total volume of 25 μL using Taq DNA Polymerase (Invitrogen) following manufacturer's protocol. ..

    Positive Control:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: .. The DNA concentration was quantified and 25 ng of input DNA (positive control), no Ab, and IPs (immunoprecipitated DNAs from anti-GFP) was analyzed by qPCR on the StepOnePlus Real-Time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). ..

    Concentration Assay:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: .. The DNA concentration was quantified and 25 ng of input DNA (positive control), no Ab, and IPs (immunoprecipitated DNAs from anti-GFP) was analyzed by qPCR on the StepOnePlus Real-Time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). ..

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue
    Article Snippet: .. These tests include 1) Agilent Bioanalyzer of the input DNA ( ); 2) Qubit fluorometric assay (Thermo Fisher Scientific) concentration measurement of the input DNA and ChIP DNA product; and 3) qPCR of the input DNA and ChIP DNA product ( ). .. First, successful chromatin digestion, resulting in an overwhelming share of mononucleosomes, should be confirmed using Agilent Bioanalyzer (the trace will show the major DNA fragment around 150 bp) ( ).

    Purification:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: .. Purified DNA from immunoprecipitates and from input DNA was analyzed by quantitative real-time PCR on an Applied Biosystems 7500 Real-Time PCR System using Power SYBR® Green PCR Master Mix (Applied Biosystems). ..

    SYBR Green Assay:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: .. The DNA concentration was quantified and 25 ng of input DNA (positive control), no Ab, and IPs (immunoprecipitated DNAs from anti-GFP) was analyzed by qPCR on the StepOnePlus Real-Time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). ..

    Article Title: The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency *
    Article Snippet: .. Quantitative real-time PCR was performed on input DNA and antibody-specific ChIP DNA using SYBR Green Master Mix reagents with an ABI Step one plus thermocycler (Applied Biosystems, Foster City, CA) detection system. .. Enrichment of RB family members at target gene promoters was examined using primers spanning known E2F binding sites at the murine CCNA2 and MCM10 loci.

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: .. Purified DNA from immunoprecipitates and from input DNA was analyzed by quantitative real-time PCR on an Applied Biosystems 7500 Real-Time PCR System using Power SYBR® Green PCR Master Mix (Applied Biosystems). ..

    Immunoprecipitation:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: .. The DNA concentration was quantified and 25 ng of input DNA (positive control), no Ab, and IPs (immunoprecipitated DNAs from anti-GFP) was analyzed by qPCR on the StepOnePlus Real-Time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). ..

    Article Title: BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation
    Article Snippet: .. Both immunoprecipitated DNA and input DNA were analyzed by real-time quantitative PCR (qPCR; Applied Biosystems). .. Monoclonal anti-myc antibody (Sigma-Aldrich) was used.

    Polymerase Chain Reaction:

    Article Title: The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism
    Article Snippet: .. The DNA concentration was quantified and 25 ng of input DNA (positive control), no Ab, and IPs (immunoprecipitated DNAs from anti-GFP) was analyzed by qPCR on the StepOnePlus Real-Time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). ..

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: .. Purified DNA from immunoprecipitates and from input DNA was analyzed by quantitative real-time PCR on an Applied Biosystems 7500 Real-Time PCR System using Power SYBR® Green PCR Master Mix (Applied Biosystems). ..

    Article Title: BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer
    Article Snippet: .. PCR reactions for amplification of chromatin immunoprecipitates and input DNA were performed in a total volume of 25 μL using Taq DNA Polymerase (Invitrogen) following manufacturer's protocol. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication
    Article Snippet: .. To eliminate the transfected input DNA, the RNA preparation was further treated with 2 U RNase-free recombinant DNase I for 30 minutes at 37°C by using the DNA-free Kit (Ambion), followed by re-suspension in RNase-free H2 O. RT-PCR was employed to detect the (-) gRNAs and sg mRNAs using specific primers. ..

    Produced:

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells
    Article Snippet: .. The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific). .. The purified and digested DNA was resolved on a 0.8% TAE-agarose gel, denatured and transferred to a Hybond-N+ filter (Amersham Pharmacia Biotech).

    Recombinant:

    Article Title: A 5'-proximal Stem-loop Structure of 5' Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus Genome Is Key for Virus Replication
    Article Snippet: .. To eliminate the transfected input DNA, the RNA preparation was further treated with 2 U RNase-free recombinant DNase I for 30 minutes at 37°C by using the DNA-free Kit (Ambion), followed by re-suspension in RNase-free H2 O. RT-PCR was employed to detect the (-) gRNAs and sg mRNAs using specific primers. ..

    Chromatin Immunoprecipitation:

    Article Title: The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency *
    Article Snippet: .. Quantitative real-time PCR was performed on input DNA and antibody-specific ChIP DNA using SYBR Green Master Mix reagents with an ABI Step one plus thermocycler (Applied Biosystems, Foster City, CA) detection system. .. Enrichment of RB family members at target gene promoters was examined using primers spanning known E2F binding sites at the murine CCNA2 and MCM10 loci.

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue
    Article Snippet: .. These tests include 1) Agilent Bioanalyzer of the input DNA ( ); 2) Qubit fluorometric assay (Thermo Fisher Scientific) concentration measurement of the input DNA and ChIP DNA product; and 3) qPCR of the input DNA and ChIP DNA product ( ). .. First, successful chromatin digestion, resulting in an overwhelming share of mononucleosomes, should be confirmed using Agilent Bioanalyzer (the trace will show the major DNA fragment around 150 bp) ( ).

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    Thermo Fisher input dna
    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic <t>DNA</t> was extracted 3 and 5 days after the transfection, linearized with <t>BglI</t> and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
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    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

    Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Journal: Biological psychiatry

    Article Title: Practical Guidelines for High-Resolution Epigenomic Profiling of Nucleosomal Histones in Postmortem Human Brain Tissue

    doi: 10.1016/j.biopsych.2016.03.1048

    Figure Lengend Snippet: Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) quality controls (QC). Quality controls include: (A) visual inspection and quantification of nuclei separated into neuronal (NeuN+) and nonneuronal (NeuN−) fraction by fluorescence-activated (cell) sorting of nuclei, including linear correlation of nuclei number with approximate prefrontal cortex gray matter tissue weight, as indicated. (B) DNA agarose gel from native chromatin digested with different amounts of micrococcal nuclease (MNase). The predominant ~150 base pair (bp) band confirms that the bulk of chromatin has been digested into mononucleosomes. (C) ). Note no cross-reactivity other than weak activity against the dimethylated form, H3K4me2. (D) Agilent Bioanalyzer QC after ChIP confirms that predominant portion of pulldown was comprised by mononucleosomes as evidenced by sharp peak at ~148 bp. (E) ChIP-quantitative polymerase chain reaction (qPCR) confirms H3K4me3 enrichment in neuronal NeuN+ nuclei fraction (blue curve) and nonneuronal NeuN− nuclei fraction (red curve) for neuronal gene GRIN2B (upper panel) but not for negative control HBB globin sequences (lower panel). Note that the input DNA qPCR signals (dark and light green curves) are similar for these two genes. (F) Agilent Bioanalyzer QC after library preparation, confirming that large majority of DNA molecules locate to 275 bp, representing correct library ligation product (see text). (G) Early bioinformatical analyses include FASTQC, BWA, and other established programs. Note consistent GRIN2B H3K4me3 enrichment observed in the NeuN+ and NeuN− ChIP-seq tracks visualized in Integrative Genomics Viewer (IGV) browser when compared with the corresponding ChIP-qPCR signals above (E) (see text for more details). (H) FASTQC analysis of raw ChIP-seq data, represented here as the sequence quality score (y axis) versus base pair position (x axis), is an important initial step in ChIP-seq data quality control. The graph background colors separate the y axis into very good quality calls (green, score > 28), calls of reasonable quality (orange, score = 20–28), and calls of poor quality (red, score

    Article Snippet: These tests include 1) Agilent Bioanalyzer of the input DNA ( ); 2) Qubit fluorometric assay (Thermo Fisher Scientific) concentration measurement of the input DNA and ChIP DNA product; and 3) qPCR of the input DNA and ChIP DNA product ( ).

    Techniques: Chromatin Immunoprecipitation, Sequencing, Fluorescence, FACS, Agarose Gel Electrophoresis, Activity Assay, Real-time Polymerase Chain Reaction, Negative Control, Ligation

    Effect Of The Variant On OASIS Function. A) IVT. The variant protein (VT312) is stably expressed and does not appear truncated compared to wild type (WT) protein. IVT with no DNA input or with a construct bearing GFP DNA were included as (−) and (+) controls for in vitro transcription, respectively. Western Blot. B) The family variant causes a defect in DNA binding. A shift is seen when IVT WT OASIS is incubated with the radiolabeled oligo containing the UPRE-like sequence (lane 1). This shift is competed away when unlabeled oligo is added (lane 2). The shift is absent when radiolabeled oligo is incubated with IVT VT312 OASIS (lane 3). Background bands are likely binding interactions between labeled oligo and proteins from the HeLa lysate used to perform IVT. EMSA.

    Journal: Genetics in medicine : official journal of the American College of Medical Genetics

    Article Title: Monoallelic and Biallelic CREB3L1 Variant Causes Mild and Severe Osteogenesis Imperfecta, Respectively

    doi: 10.1038/gim.2017.115

    Figure Lengend Snippet: Effect Of The Variant On OASIS Function. A) IVT. The variant protein (VT312) is stably expressed and does not appear truncated compared to wild type (WT) protein. IVT with no DNA input or with a construct bearing GFP DNA were included as (−) and (+) controls for in vitro transcription, respectively. Western Blot. B) The family variant causes a defect in DNA binding. A shift is seen when IVT WT OASIS is incubated with the radiolabeled oligo containing the UPRE-like sequence (lane 1). This shift is competed away when unlabeled oligo is added (lane 2). The shift is absent when radiolabeled oligo is incubated with IVT VT312 OASIS (lane 3). Background bands are likely binding interactions between labeled oligo and proteins from the HeLa lysate used to perform IVT. EMSA.

    Article Snippet: IVT with no DNA input or with a construct bearing GFP DNA (pCFE-GFP; ThermoFisher Scientific, Waltham, MA, USA) were included as (−) and (+) controls, respectively, for IVT.

    Techniques: Variant Assay, Stable Transfection, Construct, In Vitro, Western Blot, Binding Assay, Incubation, Sequencing, Labeling