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    Millipore total cellular dna
    Detection of Fragmented <t>DNA</t> in FB1-Treated Protoplasts. Protoplasts were prepared from wild-type plants and incubated for 6 hr in the light in the presence of 0.001% methanol or 70 nM FB1. DNA molecules in the protoplasts then were stained with <t>Hoechst</t> 33342, and free 3′-OH groups in the molecules were labeled by the TUNEL technique as described in Methods. (A) Hoechst 33342 staining of methanol-treated protoplasts. (B) TUNEL staining of methanol-treated protoplasts. (C) Hoechst 33342 staining of FB1-treated protoplasts. (D) TUNEL staining of FB1-treated protoplasts.
    Total Cellular Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore total dna
    Detection of <t>DNA</t> fragmentaion in OTA-treated Arabidopsis protoplasts. ( A–C ) Nuclear apoptotic alterations in Arabidopsis protoplasts treated with 0.01 mM OTA for 10 h. Protoplasts labeled with the <t>Hoechst</t> 33342 (6.25 µg/mL) and observed under UV light excitation. ( A ) Control: Normal protoplast. ( B ) Cell deformed, swelled and chambered. ( C ) Chromatin condensed and apoptotic body formed (1000×). ( D–F ) Example of TUNEL-positive nuclei in OTA treated Arabidopsis protoplasts incubated with 0.01 mM OTA for 10 h. Protoplasts labeled with the TUNEL reaction observed under blue light excitation ( D ) Surviving protoplasts. ( E,F ) Protoplasts labeled with TUNEL reaction observed under blue light excitation. White arrow indicate TUNEL-positive nucleus (600×). ( G ) The percentage of TUNEL-positive nuclei in different treatment. For each replicate at least 100 protoplasts were evaluated. The mean value was calculated by three replicates, and the standard deviation was marked in the vertical bars.
    Total Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore total urine dna
    Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with <t>DNA</t> Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the <t>microarrays.</t>
    Total Urine Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore total dna extraction
    Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with <t>DNA</t> Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the <t>microarrays.</t>
    Total Dna Extraction, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Detection of Fragmented DNA in FB1-Treated Protoplasts. Protoplasts were prepared from wild-type plants and incubated for 6 hr in the light in the presence of 0.001% methanol or 70 nM FB1. DNA molecules in the protoplasts then were stained with Hoechst 33342, and free 3′-OH groups in the molecules were labeled by the TUNEL technique as described in Methods. (A) Hoechst 33342 staining of methanol-treated protoplasts. (B) TUNEL staining of methanol-treated protoplasts. (C) Hoechst 33342 staining of FB1-treated protoplasts. (D) TUNEL staining of FB1-treated protoplasts.

    Journal: The Plant Cell

    Article Title: Fumonisin B1-Induced Cell Death in Arabidopsis Protoplasts Requires Jasmonate-, Ethylene-, and Salicylate-Dependent Signaling Pathways

    doi:

    Figure Lengend Snippet: Detection of Fragmented DNA in FB1-Treated Protoplasts. Protoplasts were prepared from wild-type plants and incubated for 6 hr in the light in the presence of 0.001% methanol or 70 nM FB1. DNA molecules in the protoplasts then were stained with Hoechst 33342, and free 3′-OH groups in the molecules were labeled by the TUNEL technique as described in Methods. (A) Hoechst 33342 staining of methanol-treated protoplasts. (B) TUNEL staining of methanol-treated protoplasts. (C) Hoechst 33342 staining of FB1-treated protoplasts. (D) TUNEL staining of FB1-treated protoplasts.

    Article Snippet: Total cellular DNA was stained with Hoechst 33342 (Sigma; 5 μg mL−1 in PBS), and the free 3′-OH groups in the DNA were labeled by the TUNEL method, using the In Situ Cell Death Detection Kit (Boehringer Mannheim) as instructed by the manufacturer (see also ).

    Techniques: Incubation, Staining, Labeling, TUNEL Assay

    Orc1 function regulates replication fork progression. (A) WT and orc1-161 cells were synchronized with α-factor for 3 h at 23°C, shifted to 32°C for 1 h, and released from α-factor at 32°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Immunoblot analysis of phosphorylated Rad53 (Rad53-P); both rows are from the same blot and exposure. Molecular weight markers were not run on this gel; for the migration of size markers relative to the bands detected by this antibody, see Fig. 2 F . Black line indicates that intervening lanes have been spliced out. (D) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (E) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in D, and were calculated from both replicates.

    Journal: The Journal of Cell Biology

    Article Title: The level of origin firing inversely affects the rate of replication fork progression

    doi: 10.1083/jcb.201208060

    Figure Lengend Snippet: Orc1 function regulates replication fork progression. (A) WT and orc1-161 cells were synchronized with α-factor for 3 h at 23°C, shifted to 32°C for 1 h, and released from α-factor at 32°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Immunoblot analysis of phosphorylated Rad53 (Rad53-P); both rows are from the same blot and exposure. Molecular weight markers were not run on this gel; for the migration of size markers relative to the bands detected by this antibody, see Fig. 2 F . Black line indicates that intervening lanes have been spliced out. (D) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (E) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in D, and were calculated from both replicates.

    Article Snippet: Immunoprecipitated and total DNA samples (from BrdU-IP-chip and ChIP-chip) were amplified using WGA2 (Sigma-Aldrich), labeled with Cy5 and Cy3, respectively, and hybridized to custom-designed oligonucleotide-based tiling microarrays (Roche) using the hybridization system (Maui; Roche) according to the manufacturer’s instructions; further details are provided in and , ).

    Techniques: Molecular Weight, Migration, BrdU Incorporation Assay, Chromatin Immunoprecipitation

    Deregulated origin firing in mec1-100 slows replication forks. (A) WT and mec1-100 cells were synchronized with α-factor for 4 h at 23°C and released from α-factor at 23°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Immunoblot analysis of phosphorylated Rad53 (Rad53-P); both rows are from the same blot and exposure. Molecular weight markers were not run on this gel; for the migration of size markers relative to the bands detected by this antibody, see Fig. 2 F . (D) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (E) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for entire chromosome VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in D, and were calculated from both replicates.

    Journal: The Journal of Cell Biology

    Article Title: The level of origin firing inversely affects the rate of replication fork progression

    doi: 10.1083/jcb.201208060

    Figure Lengend Snippet: Deregulated origin firing in mec1-100 slows replication forks. (A) WT and mec1-100 cells were synchronized with α-factor for 4 h at 23°C and released from α-factor at 23°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Immunoblot analysis of phosphorylated Rad53 (Rad53-P); both rows are from the same blot and exposure. Molecular weight markers were not run on this gel; for the migration of size markers relative to the bands detected by this antibody, see Fig. 2 F . (D) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (E) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for entire chromosome VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in D, and were calculated from both replicates.

    Article Snippet: Immunoprecipitated and total DNA samples (from BrdU-IP-chip and ChIP-chip) were amplified using WGA2 (Sigma-Aldrich), labeled with Cy5 and Cy3, respectively, and hybridized to custom-designed oligonucleotide-based tiling microarrays (Roche) using the hybridization system (Maui; Roche) according to the manufacturer’s instructions; further details are provided in and , ).

    Techniques: Molecular Weight, Migration, BrdU Incorporation Assay, Chromatin Immunoprecipitation

    Cdc7 function regulates replication fork progression. (A) WT and cdc7-as3 cells were synchronized with α-factor for 3 h and 35 min, treated with PP1 for 25 min, and released from α-factor with PP1 and with or without 0.033% MMS. (B) DNA content analysis by FACScan. Analysis of PP1 untreated cells is also shown. (C) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (D) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in C, and were calculated from both replicates.

    Journal: The Journal of Cell Biology

    Article Title: The level of origin firing inversely affects the rate of replication fork progression

    doi: 10.1083/jcb.201208060

    Figure Lengend Snippet: Cdc7 function regulates replication fork progression. (A) WT and cdc7-as3 cells were synchronized with α-factor for 3 h and 35 min, treated with PP1 for 25 min, and released from α-factor with PP1 and with or without 0.033% MMS. (B) DNA content analysis by FACScan. Analysis of PP1 untreated cells is also shown. (C) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (D) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. Data shown are from a single representative experiment out of two replicates, except data in C, and were calculated from both replicates.

    Article Snippet: Immunoprecipitated and total DNA samples (from BrdU-IP-chip and ChIP-chip) were amplified using WGA2 (Sigma-Aldrich), labeled with Cy5 and Cy3, respectively, and hybridized to custom-designed oligonucleotide-based tiling microarrays (Roche) using the hybridization system (Maui; Roche) according to the manufacturer’s instructions; further details are provided in and , ).

    Techniques: BrdU Incorporation Assay, Chromatin Immunoprecipitation

    Cdc7 functions upstream of Rad53 in fork regulation. (A) WT , cdc7-1 mcm5-bob1 , and cdc7-1 mcm5-bob1 pph3Δ cells were synchronized with α-factor for 3 h at 23°C, shifted to 32°C for 1 h, and released from α-factor at 32°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (D) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. (E) WT and cdc7-1 mcm5-bob1 cells expressing Rfa1-Myc18 were treated as in A and analyzed by ChIP-chip 35 min after release; plots are color coded as in D. (F) Immunoblot analysis of unphosphorylated (single asterisks) and phosphorylated Rad53 (double asterisk); molecular mass markers were visualized with Ponceau S. Black line indicates that intervening lanes have been spliced out. Data shown are from a single representative experiment out of two replicates, except data in C, and were calculated from both replicates.

    Journal: The Journal of Cell Biology

    Article Title: The level of origin firing inversely affects the rate of replication fork progression

    doi: 10.1083/jcb.201208060

    Figure Lengend Snippet: Cdc7 functions upstream of Rad53 in fork regulation. (A) WT , cdc7-1 mcm5-bob1 , and cdc7-1 mcm5-bob1 pph3Δ cells were synchronized with α-factor for 3 h at 23°C, shifted to 32°C for 1 h, and released from α-factor at 32°C with 0.033% MMS. (B) DNA content analysis by FACScan. (C) Heat maps of BrdU incorporation levels at origins are arranged according to each origin’s published replication timing from early to late (left to right). (D) Aliquots were pulsed with BrdU for the indicated intervals and analyzed by BrdU-IP-chip. Results for segments of chromosomes III and VI are plotted, with origin locations indicated above. (E) WT and cdc7-1 mcm5-bob1 cells expressing Rfa1-Myc18 were treated as in A and analyzed by ChIP-chip 35 min after release; plots are color coded as in D. (F) Immunoblot analysis of unphosphorylated (single asterisks) and phosphorylated Rad53 (double asterisk); molecular mass markers were visualized with Ponceau S. Black line indicates that intervening lanes have been spliced out. Data shown are from a single representative experiment out of two replicates, except data in C, and were calculated from both replicates.

    Article Snippet: Immunoprecipitated and total DNA samples (from BrdU-IP-chip and ChIP-chip) were amplified using WGA2 (Sigma-Aldrich), labeled with Cy5 and Cy3, respectively, and hybridized to custom-designed oligonucleotide-based tiling microarrays (Roche) using the hybridization system (Maui; Roche) according to the manufacturer’s instructions; further details are provided in and , ).

    Techniques: BrdU Incorporation Assay, Chromatin Immunoprecipitation, Expressing

    PCR detection of the LINE1-c- myc gene rearrangement of CTVT origin from cell-derived FNA samples. PCR products (550 bp) were resolved on a 1.5% (w/v) agarose gel. (Lane 1=100 bp DNA marker, Lane 2=positive control, Lane 3=negative control, Lanes 4–9 are samples from the vaginal mass (case 1), skin mass (case 2), nasal mass (case 7), nasal mass (case 8), nasal mass (case 11 in Table 2 ) and chronic inflammation tissue (case 9), respectively.)

    Journal: The Journal of Veterinary Medical Science

    Article Title: Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis

    doi: 10.1292/jvms.15-0710

    Figure Lengend Snippet: PCR detection of the LINE1-c- myc gene rearrangement of CTVT origin from cell-derived FNA samples. PCR products (550 bp) were resolved on a 1.5% (w/v) agarose gel. (Lane 1=100 bp DNA marker, Lane 2=positive control, Lane 3=negative control, Lanes 4–9 are samples from the vaginal mass (case 1), skin mass (case 2), nasal mass (case 7), nasal mass (case 8), nasal mass (case 11 in Table 2 ) and chronic inflammation tissue (case 9), respectively.)

    Article Snippet: The LINE1-c-myc PCR assay : Total genomic DNA was extracted from FNA-derived cells and fresh tissue using Mammalian genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO, U.S.A.) following the manufacturer’s instruction.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Agarose Gel Electrophoresis, Marker, Positive Control, Negative Control

    Phylogenetic tree of the LINE-c- myc CTVT sequence of this study and the related sequences from the Genbank database. CTVT from Thailand sequences: FNA sample (triangle) sequences of KU680469 (case 1), KU680470 (case 2), KU680471 (case 7), KU680472 (case 11 in Table 2 ) and KU680473 (case 8); and fresh tissue samples (circle) from KU680474 (case 4), KU680475 (case 5, skin mass), KU680476 (case 5, penile mass), KU680477 (case 6) and KU680478 (case 10). CTVT samples in Genbank: Canis lupus familiaris LINE-1 elememt DNA partial sequence (AB012217), LINE/c- myc junction sequence (S55298), Canis familiaris c- myc gene partial sequence (AY032723), Dog c- myc oncogene DNA with a retroposon insertion target sequence (M37386) and Dog c- myc oncogene with an inserted retroposon (M37385).

    Journal: The Journal of Veterinary Medical Science

    Article Title: Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis

    doi: 10.1292/jvms.15-0710

    Figure Lengend Snippet: Phylogenetic tree of the LINE-c- myc CTVT sequence of this study and the related sequences from the Genbank database. CTVT from Thailand sequences: FNA sample (triangle) sequences of KU680469 (case 1), KU680470 (case 2), KU680471 (case 7), KU680472 (case 11 in Table 2 ) and KU680473 (case 8); and fresh tissue samples (circle) from KU680474 (case 4), KU680475 (case 5, skin mass), KU680476 (case 5, penile mass), KU680477 (case 6) and KU680478 (case 10). CTVT samples in Genbank: Canis lupus familiaris LINE-1 elememt DNA partial sequence (AB012217), LINE/c- myc junction sequence (S55298), Canis familiaris c- myc gene partial sequence (AY032723), Dog c- myc oncogene DNA with a retroposon insertion target sequence (M37386) and Dog c- myc oncogene with an inserted retroposon (M37385).

    Article Snippet: The LINE1-c-myc PCR assay : Total genomic DNA was extracted from FNA-derived cells and fresh tissue using Mammalian genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO, U.S.A.) following the manufacturer’s instruction.

    Techniques: Sequencing

    PCR detection of the LINE1-c- myc gene from cell-derived FNA samples (cases 12 and 24, Table 2 ). PCR products (550 bp) were resolved on a 1.5% (w/v) agarose gel. (Lane1=100-bp DNA marker, Lane 2 =positive control, Lane 3 =case 12, Lane 4 =case 24 and Lane 5 =negative control)

    Journal: The Journal of Veterinary Medical Science

    Article Title: Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis

    doi: 10.1292/jvms.15-0710

    Figure Lengend Snippet: PCR detection of the LINE1-c- myc gene from cell-derived FNA samples (cases 12 and 24, Table 2 ). PCR products (550 bp) were resolved on a 1.5% (w/v) agarose gel. (Lane1=100-bp DNA marker, Lane 2 =positive control, Lane 3 =case 12, Lane 4 =case 24 and Lane 5 =negative control)

    Article Snippet: The LINE1-c-myc PCR assay : Total genomic DNA was extracted from FNA-derived cells and fresh tissue using Mammalian genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO, U.S.A.) following the manufacturer’s instruction.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Agarose Gel Electrophoresis, Marker, Positive Control, Negative Control

    Detection of DNA fragmentaion in OTA-treated Arabidopsis protoplasts. ( A–C ) Nuclear apoptotic alterations in Arabidopsis protoplasts treated with 0.01 mM OTA for 10 h. Protoplasts labeled with the Hoechst 33342 (6.25 µg/mL) and observed under UV light excitation. ( A ) Control: Normal protoplast. ( B ) Cell deformed, swelled and chambered. ( C ) Chromatin condensed and apoptotic body formed (1000×). ( D–F ) Example of TUNEL-positive nuclei in OTA treated Arabidopsis protoplasts incubated with 0.01 mM OTA for 10 h. Protoplasts labeled with the TUNEL reaction observed under blue light excitation ( D ) Surviving protoplasts. ( E,F ) Protoplasts labeled with TUNEL reaction observed under blue light excitation. White arrow indicate TUNEL-positive nucleus (600×). ( G ) The percentage of TUNEL-positive nuclei in different treatment. For each replicate at least 100 protoplasts were evaluated. The mean value was calculated by three replicates, and the standard deviation was marked in the vertical bars.

    Journal: Toxins

    Article Title: iTRAQ Mitoproteome Analysis Reveals Mechanisms of Programmed Cell Death in Arabidopsis thaliana Induced by Ochratoxin A

    doi: 10.3390/toxins9050167

    Figure Lengend Snippet: Detection of DNA fragmentaion in OTA-treated Arabidopsis protoplasts. ( A–C ) Nuclear apoptotic alterations in Arabidopsis protoplasts treated with 0.01 mM OTA for 10 h. Protoplasts labeled with the Hoechst 33342 (6.25 µg/mL) and observed under UV light excitation. ( A ) Control: Normal protoplast. ( B ) Cell deformed, swelled and chambered. ( C ) Chromatin condensed and apoptotic body formed (1000×). ( D–F ) Example of TUNEL-positive nuclei in OTA treated Arabidopsis protoplasts incubated with 0.01 mM OTA for 10 h. Protoplasts labeled with the TUNEL reaction observed under blue light excitation ( D ) Surviving protoplasts. ( E,F ) Protoplasts labeled with TUNEL reaction observed under blue light excitation. White arrow indicate TUNEL-positive nucleus (600×). ( G ) The percentage of TUNEL-positive nuclei in different treatment. For each replicate at least 100 protoplasts were evaluated. The mean value was calculated by three replicates, and the standard deviation was marked in the vertical bars.

    Article Snippet: Total DNA was visualized by staining with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) at 6.25 µg/mL for 5 min at dark, and then visualized with an Olympus BX51 fluorescent microscope, using an UV filter.

    Techniques: Labeling, TUNEL Assay, Incubation, Standard Deviation

    Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025181

    Figure Lengend Snippet: Assessment of the specificity of the ChIP protocol. A) Arx immunoprecipitation from N2a cells transfected with an Arx-expressing vector or a vector alone and using the Arx C-14 antibody from Santa Cruz. Detection using the anti-Arx-HD antibody revealed a major band at approximately 80 kD, which corresponds to the size of Arx protein. TE: total extracts, IP: immunoprecipitation, Ig: immunoglobulins. B) Enrichment of Ebf3 , Lmo1 and Shox2 promoter regions was assessed by qPCR using either Arx or PolII-immunoprecipitates or using no antibody and was compared to the total input. Gapdh was used as a positive control with DNA Pol II. Here, we show an example of the results obtained in a ChIP experiment with Arx C-14 antibody from Santa Cruz. The enrichment of Ebf3 , Lmo1 and Shox2 was checked similarly for each replicate experiment before DNA was applied to the microarrays.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025181

    Figure Lengend 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.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: 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).

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025181

    Figure Lengend 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).

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0025181

    Figure Lengend 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.

    Article Snippet: DNA amplification, labelling and hybridization on microarrays Equivalent amounts of immunoprecipitated chromatin and total input DNA were amplified in parallel, using a random primer method with the GenomePlex Complete Whole Genome Amplification Kit (15 cycles), according to the manufacturer's instructions (Sigma).

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