wizard genomic dna purification kit  (Promega)

 
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    Name:
    Wizard Plus SV Minipreps DNA Purification Systems
    Description:
    Spin and vacuum based methods for plasmid purification from 1 10ml cultures
    Catalog Number:
    a1330
    Price:
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    Category:
    Nucleic Acid Extraction Analysis Nucleic Acid Extraction Plasmid Purification
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    Structured Review

    Promega wizard genomic dna purification kit
    Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic <t>DNA</t> extracted from Pb <t>WT-GFP</t> and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P
    Spin and vacuum based methods for plasmid purification from 1 10ml cultures
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    Images

    1) Product Images from "Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands"

    Article Title: Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands

    Journal: mSphere

    doi: 10.1128/mSphere.00325-20

    Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P
    Figure Legend Snippet: Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P

    Techniques Used: Transgenic Assay, Southern Blot, Agarose Gel Electrophoresis, Hybridization, Marker, Derivative Assay, Infection, Expressing

    2) Product Images from "Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System"

    Article Title: Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026414

    Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells. ( A ) Schematic diagram of virus-activated innate antiviral signaling pathways. ( B ) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. ( C ) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.
    Figure Legend Snippet: Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells. ( A ) Schematic diagram of virus-activated innate antiviral signaling pathways. ( B ) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. ( C ) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.

    Techniques Used: Activation Assay, Expressing, Transfection, Activity Assay

    3) Product Images from "REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS"

    Article Title: REGULATION OF EXPRESSION OF STROMAL-DERIVED FACTOR-1 (SDF-1) RECEPTORS: CXCR4 AND CXCR7 IN HUMAN RHADOMYOSARCOMAS

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-09-0259

    CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p
    Figure Legend Snippet: CXCR4 promoter deletion studies and transcription binding sites analysis Panel A. Constructed CXCR4 promoter inserts. The promoter region of the CXCR4 gene from −2,237 to +62 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 was sequentially shortened according to the positions of PAX3 binding sites, and NRF-1 binding sites as depicted in the theoretical model of the cloned sequences of the promoter. The position of PAX3-, NRF-1- HRE-, NF-κb and YY1 binding sites as well as P1-3 primer pairs used in the ChIP experiment are shown. Panel B. CXCR4 promoter activity studies. RD and RD/PAX3-FKHR cells were transfected with the appropriate plasmids. Cultured cells were harvested after 24 hours and assayed for the amount of luciferase activity. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag1–7 p

    Techniques Used: Binding Assay, Construct, Clone Assay, Chromatin Immunoprecipitation, Activity Assay, Transfection, Cell Culture, Luciferase, Plasmid Preparation

    CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p
    Figure Legend Snippet: CXCR7 promoter deletion studies and transcription binding sites analysis Panel A. CXCR7 promoter inserts. The promoter region of the CXCR7 gene from −2,409 to +89 relative to the start of transcription was cloned and is described as Fragment 1. Fragment 1 shorter derivatives were obtained according to the positions of NRF-1, HRE binding sites, and NF-κB binding sites. The individual constructs were cloned to pGL4.10 vector, transfected into either RD or RD/PAX3-FKHR cells, and used in Dual Luciferase assays. (X) – mutated NF-κB and HRE binding sites. Primer pairs used in the ChIP experiment (N1–N5) flanked 5 different potential binding sites for NF-κB transcription factor. Panel B. NF-κB as a crucial transcription factor that drives CXCR7 promoter activity. Activity of particular CXCR7 promoter constructs was assayed by Dual Luciferase showing an important role of NF-κB transcription factors. Activity was measured on the basis of firefly/ Renillla luciferase activity and then the equimolar fold of difference was counted. Results are expressed relative to a value of 1.0 for cells transfected with pGL4.72 vector and empty pGL4.10. Averages of duplicates from three independent experiments are shown. Values are given as the mean ± SEM. Frag 1–6 p

    Techniques Used: Binding Assay, Clone Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Chromatin Immunoprecipitation, Activity Assay

    4) Product Images from "Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿"

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01135-10

    Restriction digest patterns electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. (A) FnpΦ02 genomic DNA digested with restriction enzymes. Lanes: L1, 100-bp DNA ladder marker (Fermentas Inc., Canada); L2, 1-kb ladder marker (Fermentas Inc., Canada); L3, undigested DNA; L4, DNA/HindIII; L5, DNA/DraI; L6, DNA/XbaI; and L7, lambda DNA/HindIII. (B) Higher magnification of the bands of the digestion of FnpΦ02 with HindIII used for genome size determination. Selected sizes of the marker are indicated in panel A.
    Figure Legend Snippet: Restriction digest patterns electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. (A) FnpΦ02 genomic DNA digested with restriction enzymes. Lanes: L1, 100-bp DNA ladder marker (Fermentas Inc., Canada); L2, 1-kb ladder marker (Fermentas Inc., Canada); L3, undigested DNA; L4, DNA/HindIII; L5, DNA/DraI; L6, DNA/XbaI; and L7, lambda DNA/HindIII. (B) Higher magnification of the bands of the digestion of FnpΦ02 with HindIII used for genome size determination. Selected sizes of the marker are indicated in panel A.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Marker, Lambda DNA Preparation

    5) Product Images from "Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands"

    Article Title: Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands

    Journal: mSphere

    doi: 10.1128/mSphere.00325-20

    Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P
    Figure Legend Snippet: Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P

    Techniques Used: Transgenic Assay, Southern Blot, Agarose Gel Electrophoresis, Hybridization, Marker, Derivative Assay, Infection, Expressing

    Repression of ron4 or ron5 expression decreases sporozoite invasion efficiency for mosquito salivary glands. Sporozoite numbers collected and counted from midguts, hemolymph, and salivary glands of transgenic parasite-infected mosquitoes at days 23 to 26 postfeeding. Each dot represents the average sporozoite numbers from 20 to 30 mosquitoes. The horizontal bars and error bars indicate the means and standard deviation from at least four independent experiments using independently prepared mosquito groups. (A) The numbers of sporozoites of RON4-cont, RON4-cKD cl1, and RON4-cKD cl2 are shown as dot plots. P values were calculated by using the Kruskal-Wallis test. No significant difference was observed in the number of sporozoites collected from midguts ( P value: 0.7171, indicated as ns) or from hemolymph ( P value: 0.4689, indicated as ns). In contrast, the number of sporozoites collected from salivary glands was significantly different ( P value: 0.0001). The P value from post hoc analysis, the Dunn multiple-comparison test, is shown in the graph (**, P
    Figure Legend Snippet: Repression of ron4 or ron5 expression decreases sporozoite invasion efficiency for mosquito salivary glands. Sporozoite numbers collected and counted from midguts, hemolymph, and salivary glands of transgenic parasite-infected mosquitoes at days 23 to 26 postfeeding. Each dot represents the average sporozoite numbers from 20 to 30 mosquitoes. The horizontal bars and error bars indicate the means and standard deviation from at least four independent experiments using independently prepared mosquito groups. (A) The numbers of sporozoites of RON4-cont, RON4-cKD cl1, and RON4-cKD cl2 are shown as dot plots. P values were calculated by using the Kruskal-Wallis test. No significant difference was observed in the number of sporozoites collected from midguts ( P value: 0.7171, indicated as ns) or from hemolymph ( P value: 0.4689, indicated as ns). In contrast, the number of sporozoites collected from salivary glands was significantly different ( P value: 0.0001). The P value from post hoc analysis, the Dunn multiple-comparison test, is shown in the graph (**, P

    Techniques Used: Expressing, Transgenic Assay, Infection, Standard Deviation

    RON2 and RON4 reciprocally affect trafficking to rhoptries in oocyst-derived sporozoites. (A) Western blot analysis of RON complex components in RON2-cKD, RON4-cKD, and RON5-cKD oocyst-derived sporozoites. To investigate the effect of gene knockdowns on rhoptry proteins, homogenates from 100,000 oocyst-derived sporozoites were separated by SDS-PAGE and RON2, RON4, RON5, and RAMA proteins were detected using specific antibodies. TRAP was also detected as a micronemal protein involved in sporozoite motility. The relative intensities of each band were normalized by RAMA and are shown in Fig. S6A . Specific bands corresponding to the full-length and processed forms are indicated by open and closed arrowheads, respectively. (B) Rhoptry neck protein localization analyses in Pb WT-GFP, RON2-cKD, RON4-cKD, and RON5-cKD oocyst-derived sporozoites. Sporozoites collected from midguts were fixed by acetone on glass slides and incubated with specific antibodies against RON2, RON4, RON5, or RAMA, followed by incubation with secondary antibodies conjugated to Alexa 488 (green). Nuclei are visualized with DAPI (blue). Bar, 5 μm. Additional images are shown in Fig. S7 . (C) Immunoelectron microscopy analyses of RON2, RON4, and RON12 localization in transgenic oocyst-derived sporozoites. RON2 and RON4 localizations to rhoptries were perturbed by RON4 and RON2 knockdown but not by RON5 knockdown. RON12 localization is not affected by repression of any components of the RON complex (lower panels). Bars, 500 nm.
    Figure Legend Snippet: RON2 and RON4 reciprocally affect trafficking to rhoptries in oocyst-derived sporozoites. (A) Western blot analysis of RON complex components in RON2-cKD, RON4-cKD, and RON5-cKD oocyst-derived sporozoites. To investigate the effect of gene knockdowns on rhoptry proteins, homogenates from 100,000 oocyst-derived sporozoites were separated by SDS-PAGE and RON2, RON4, RON5, and RAMA proteins were detected using specific antibodies. TRAP was also detected as a micronemal protein involved in sporozoite motility. The relative intensities of each band were normalized by RAMA and are shown in Fig. S6A . Specific bands corresponding to the full-length and processed forms are indicated by open and closed arrowheads, respectively. (B) Rhoptry neck protein localization analyses in Pb WT-GFP, RON2-cKD, RON4-cKD, and RON5-cKD oocyst-derived sporozoites. Sporozoites collected from midguts were fixed by acetone on glass slides and incubated with specific antibodies against RON2, RON4, RON5, or RAMA, followed by incubation with secondary antibodies conjugated to Alexa 488 (green). Nuclei are visualized with DAPI (blue). Bar, 5 μm. Additional images are shown in Fig. S7 . (C) Immunoelectron microscopy analyses of RON2, RON4, and RON12 localization in transgenic oocyst-derived sporozoites. RON2 and RON4 localizations to rhoptries were perturbed by RON4 and RON2 knockdown but not by RON5 knockdown. RON12 localization is not affected by repression of any components of the RON complex (lower panels). Bars, 500 nm.

    Techniques Used: Derivative Assay, Western Blot, SDS Page, Incubation, Immuno-Electron Microscopy, Transgenic Assay

    RON4 and RON5 are important for hemolymph sporozoite attachment. (A) The sporozoite motility patterns on glass slides for RON2-cKD, RON4-cKD, and RON5-cKD transgenic parasites were compared with that of Pb WT-GFP. Sporozoites collected from hemolymph of infected mosquitoes were activated by incubation in 10% FCS-containing medium, and their movement patterns were classified into three categories: gliding, waving, and drifting. At least 75 hemolymph sporozoites were observed for each parasite line, and the mean proportions showing each moving pattern from five independent experiments are shown as a bar graph with standard deviations. P values were calculated by the one-way ANOVA test with the Tukey multiple-comparison test (****, P
    Figure Legend Snippet: RON4 and RON5 are important for hemolymph sporozoite attachment. (A) The sporozoite motility patterns on glass slides for RON2-cKD, RON4-cKD, and RON5-cKD transgenic parasites were compared with that of Pb WT-GFP. Sporozoites collected from hemolymph of infected mosquitoes were activated by incubation in 10% FCS-containing medium, and their movement patterns were classified into three categories: gliding, waving, and drifting. At least 75 hemolymph sporozoites were observed for each parasite line, and the mean proportions showing each moving pattern from five independent experiments are shown as a bar graph with standard deviations. P values were calculated by the one-way ANOVA test with the Tukey multiple-comparison test (****, P

    Techniques Used: Transgenic Assay, Infection, Incubation

    6) Product Images from "The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression ▿"

    Article Title: The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01265-07

    FOXO3a induces the expression of the human PIK3CA gene through a promoter region upstream of a novel 5′ exon. (A) Mapping of the human p110α transcription start site using 5′-RACE. The total RNA prepared from K562-FOXO3a(A3):ER cells after 24 h of 4-OHT induction was reverse transcribed, and the cDNA was subjected to two sequential PCRs using primers from the Ambion FirstChoice RLM-RACE kit and nested PCR primers specific to exon 2 of the human p110 α gene. The nested PCR products were analyzed by agarose gel electrophoresis, and the major PCR product was clearly visible as a band of about 300 bp. Molecular size markers (base pairs) are indicated on the left of the promoter sequence relative to the major transcription start site. (Bottom) Schematic representation of the 5′ region of the human PIK3CA genes. The exons (boxes), their sizes in base pairs, and their positions relative to the major transcription start site are indicated. Introns are represented by thick lines. The sequence and genome location of the novel exon 1A are shown. Boxes underneath show the relative positions of the three putative human PIK3CA promoter constructs A, B, and C. (B) K562 cells were transiently transfected with 1 μg of each of the three putative human p110 α promoter/reporter constructs, together with increasing amounts (0, 1, 5, and 10 μg) of pLPCFOXO3a-WT or -A3. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. (C) K562 cells were transiently transfected with 1 μg of each of the human PIK3CA promoter/reporter constructs, together with 0 or 5 μg of pLPCFOXO3a-A3, and processed as described above. The induction of the PIK3CA promoter by FOXO3a is shown on the right. (D) ChIP analysis of the human PIK3CA promoter. Protein-DNA complexes from K562 FOXO3a(A3):ER cells cultured in the presence or absence of 4-OHT for 4 h were subjected to immunoprecipitation with antibodies against immunoglobulin G (IgG) (nonspecific) and FOXO3a, as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels. The primer pairs used were 5′-GCTTTTTCTGCTATGACACACAACTTC-3′ and 5′-GCACCTGGCCTATTTGTGATTTTTA-3′ (positions −2000 to −1741), 5′-GTGAAACTACGACCACAAAGAGGA-3′ and 5′-AGCATCGGTTCCTCCTTGAA-3′ (positions −1127 to −946) 5′-CGCCTTCGGGATGGTATACAA-3′ and 5′-GAGGGTGTTGTGTCATCCTAGGAC-3′ (positions −548 to −251), and 5′-GACACAACACCCTCACTACTGCA-3′ and 5′-GGCAGAGCCTACAATCCCC-3′ (positions −264 to −55).
    Figure Legend Snippet: FOXO3a induces the expression of the human PIK3CA gene through a promoter region upstream of a novel 5′ exon. (A) Mapping of the human p110α transcription start site using 5′-RACE. The total RNA prepared from K562-FOXO3a(A3):ER cells after 24 h of 4-OHT induction was reverse transcribed, and the cDNA was subjected to two sequential PCRs using primers from the Ambion FirstChoice RLM-RACE kit and nested PCR primers specific to exon 2 of the human p110 α gene. The nested PCR products were analyzed by agarose gel electrophoresis, and the major PCR product was clearly visible as a band of about 300 bp. Molecular size markers (base pairs) are indicated on the left of the promoter sequence relative to the major transcription start site. (Bottom) Schematic representation of the 5′ region of the human PIK3CA genes. The exons (boxes), their sizes in base pairs, and their positions relative to the major transcription start site are indicated. Introns are represented by thick lines. The sequence and genome location of the novel exon 1A are shown. Boxes underneath show the relative positions of the three putative human PIK3CA promoter constructs A, B, and C. (B) K562 cells were transiently transfected with 1 μg of each of the three putative human p110 α promoter/reporter constructs, together with increasing amounts (0, 1, 5, and 10 μg) of pLPCFOXO3a-WT or -A3. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. (C) K562 cells were transiently transfected with 1 μg of each of the human PIK3CA promoter/reporter constructs, together with 0 or 5 μg of pLPCFOXO3a-A3, and processed as described above. The induction of the PIK3CA promoter by FOXO3a is shown on the right. (D) ChIP analysis of the human PIK3CA promoter. Protein-DNA complexes from K562 FOXO3a(A3):ER cells cultured in the presence or absence of 4-OHT for 4 h were subjected to immunoprecipitation with antibodies against immunoglobulin G (IgG) (nonspecific) and FOXO3a, as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels. The primer pairs used were 5′-GCTTTTTCTGCTATGACACACAACTTC-3′ and 5′-GCACCTGGCCTATTTGTGATTTTTA-3′ (positions −2000 to −1741), 5′-GTGAAACTACGACCACAAAGAGGA-3′ and 5′-AGCATCGGTTCCTCCTTGAA-3′ (positions −1127 to −946) 5′-CGCCTTCGGGATGGTATACAA-3′ and 5′-GAGGGTGTTGTGTCATCCTAGGAC-3′ (positions −548 to −251), and 5′-GACACAACACCCTCACTACTGCA-3′ and 5′-GGCAGAGCCTACAATCCCC-3′ (positions −264 to −55).

    Techniques Used: Expressing, Nested PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Construct, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Amplification

    7) Product Images from "The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression ▿"

    Article Title: The Forkhead Transcription Factor FOXO3a Increases Phosphoinositide-3 Kinase/Akt Activity in Drug-Resistant Leukemic Cells through Induction of PIK3CA Expression ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01265-07

    FOXO3a induces the expression of the human PIK3CA gene through a promoter region upstream of a novel 5′ exon. (A) Mapping of the human p110α transcription start site using 5′-RACE. The total RNA prepared from K562-FOXO3a(A3):ER cells after 24 h of 4-OHT induction was reverse transcribed, and the cDNA was subjected to two sequential PCRs using primers from the Ambion FirstChoice RLM-RACE kit and nested PCR primers specific to exon 2 of the human p110 α gene. The nested PCR products were analyzed by agarose gel electrophoresis, and the major PCR product was clearly visible as a band of about 300 bp. Molecular size markers (base pairs) are indicated on the left of the promoter sequence relative to the major transcription start site. (Bottom) Schematic representation of the 5′ region of the human PIK3CA genes. The exons (boxes), their sizes in base pairs, and their positions relative to the major transcription start site are indicated. Introns are represented by thick lines. The sequence and genome location of the novel exon 1A are shown. Boxes underneath show the relative positions of the three putative human PIK3CA promoter constructs A, B, and C. (B) K562 cells were transiently transfected with 1 μg of each of the three putative human p110 α promoter/reporter constructs, together with increasing amounts (0, 1, 5, and 10 μg) of pLPCFOXO3a-WT or -A3. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. (C) K562 cells were transiently transfected with 1 μg of each of the human PIK3CA promoter/reporter constructs, together with 0 or 5 μg of pLPCFOXO3a-A3, and processed as described above. The induction of the PIK3CA promoter by FOXO3a is shown on the right. (D) ChIP analysis of the human PIK3CA promoter. Protein-DNA complexes from K562 FOXO3a(A3):ER cells cultured in the presence or absence of 4-OHT for 4 h were subjected to immunoprecipitation with antibodies against immunoglobulin G (IgG) (nonspecific) and FOXO3a, as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels. The primer pairs used were 5′-GCTTTTTCTGCTATGACACACAACTTC-3′ and 5′-GCACCTGGCCTATTTGTGATTTTTA-3′ (positions −2000 to −1741), 5′-GTGAAACTACGACCACAAAGAGGA-3′ and 5′-AGCATCGGTTCCTCCTTGAA-3′ (positions −1127 to −946) 5′-CGCCTTCGGGATGGTATACAA-3′ and 5′-GAGGGTGTTGTGTCATCCTAGGAC-3′ (positions −548 to −251), and 5′-GACACAACACCCTCACTACTGCA-3′ and 5′-GGCAGAGCCTACAATCCCC-3′ (positions −264 to −55).
    Figure Legend Snippet: FOXO3a induces the expression of the human PIK3CA gene through a promoter region upstream of a novel 5′ exon. (A) Mapping of the human p110α transcription start site using 5′-RACE. The total RNA prepared from K562-FOXO3a(A3):ER cells after 24 h of 4-OHT induction was reverse transcribed, and the cDNA was subjected to two sequential PCRs using primers from the Ambion FirstChoice RLM-RACE kit and nested PCR primers specific to exon 2 of the human p110 α gene. The nested PCR products were analyzed by agarose gel electrophoresis, and the major PCR product was clearly visible as a band of about 300 bp. Molecular size markers (base pairs) are indicated on the left of the promoter sequence relative to the major transcription start site. (Bottom) Schematic representation of the 5′ region of the human PIK3CA genes. The exons (boxes), their sizes in base pairs, and their positions relative to the major transcription start site are indicated. Introns are represented by thick lines. The sequence and genome location of the novel exon 1A are shown. Boxes underneath show the relative positions of the three putative human PIK3CA promoter constructs A, B, and C. (B) K562 cells were transiently transfected with 1 μg of each of the three putative human p110 α promoter/reporter constructs, together with increasing amounts (0, 1, 5, and 10 μg) of pLPCFOXO3a-WT or -A3. Cells were harvested 24 h after transfection and assayed for luciferase activity. All relative luciferase activity values are corrected for cotransfected Renilla activity. All data shown represent the averages of data from three independent experiments, and the error bars show the standard deviations. (C) K562 cells were transiently transfected with 1 μg of each of the human PIK3CA promoter/reporter constructs, together with 0 or 5 μg of pLPCFOXO3a-A3, and processed as described above. The induction of the PIK3CA promoter by FOXO3a is shown on the right. (D) ChIP analysis of the human PIK3CA promoter. Protein-DNA complexes from K562 FOXO3a(A3):ER cells cultured in the presence or absence of 4-OHT for 4 h were subjected to immunoprecipitation with antibodies against immunoglobulin G (IgG) (nonspecific) and FOXO3a, as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the indicated primers and resolved in 2% agarose gels. The primer pairs used were 5′-GCTTTTTCTGCTATGACACACAACTTC-3′ and 5′-GCACCTGGCCTATTTGTGATTTTTA-3′ (positions −2000 to −1741), 5′-GTGAAACTACGACCACAAAGAGGA-3′ and 5′-AGCATCGGTTCCTCCTTGAA-3′ (positions −1127 to −946) 5′-CGCCTTCGGGATGGTATACAA-3′ and 5′-GAGGGTGTTGTGTCATCCTAGGAC-3′ (positions −548 to −251), and 5′-GACACAACACCCTCACTACTGCA-3′ and 5′-GGCAGAGCCTACAATCCCC-3′ (positions −264 to −55).

    Techniques Used: Expressing, Nested PCR, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, Construct, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Amplification

    8) Product Images from "Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System"

    Article Title: Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026414

    Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells. ( A ) Schematic diagram of virus-activated innate antiviral signaling pathways. ( B ) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. ( C ) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.
    Figure Legend Snippet: Comparison of IRF3 dimerization using the DLR-PD assay and activation of the IRF3 regulatory element PRDIII-I induced by different molecules in HEK293 cells. ( A ) Schematic diagram of virus-activated innate antiviral signaling pathways. ( B ) Activation of the IRF3 regulatory element PRDIII-I induced by different expression vectors (control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε) in HEK293 cells 24 h post-transfection. The IRF3 regulatory element PRDIII-I was assayed with 4xPRDIII-I-Fluc and the pRL-TK dual reporter. After being normalized by Rluc activity, Fluc activity from different cell lysates was compared with that of control pCDNA3.1-transfected cells. ( C ) Analysis of the association of HAVI-Fluc-IRF3 with Rluc-IRF3 induced by different molecules using the DLR-PD assay. HEK293 cells were co-transfected with HAVI-Fluc-IRF3 (20 ng), Rluc-IRF3 (10 ng) and different expression vectors (100 ng of control pCDNA3.1, GFP2-RIG-I, Halo-MDA5, eYFP-MAVS, Myc-TRAF3, Myc-TBK1 or Myc-IKKε). Subsequently, the DLR-PD assay was performed. The Rluc/Fluc ratio on the beads was calculated by dividing the Rluc activity by the Fluc activity measured on the beads.

    Techniques Used: Activation Assay, Expressing, Transfection, Activity Assay

    9) Product Images from "Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major)"

    Article Title: Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major)

    Journal: Archives of toxicology

    doi: 10.1007/s00204-016-1732-9

    Results of transactivation of rsAHR1, rsAHR2 and rsCYP1A1 promoter-driven reporter genes by TCDD treatment. To detect XRE independent response, the assay using promoterless-pGL4.10 was also performed. rsAHR1 (a) or rsAHR2 (b) expression vector was transfected in COS-7. Each bar represents the mean ± SD of fold induction of RLU from 3 or 4 replicates for each concentration from one independent experiments. Different letters on each bar denote significant differences between DMSO- and TCDD-treated cells ( p
    Figure Legend Snippet: Results of transactivation of rsAHR1, rsAHR2 and rsCYP1A1 promoter-driven reporter genes by TCDD treatment. To detect XRE independent response, the assay using promoterless-pGL4.10 was also performed. rsAHR1 (a) or rsAHR2 (b) expression vector was transfected in COS-7. Each bar represents the mean ± SD of fold induction of RLU from 3 or 4 replicates for each concentration from one independent experiments. Different letters on each bar denote significant differences between DMSO- and TCDD-treated cells ( p

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Concentration Assay

    10) Product Images from "Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells"

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells

    Journal: Stem Cells International

    doi: 10.1155/2019/1395301

    Persistence of syngeneic MSCs in immune-competent and immune-deficient animal models. (a) NOD ( n = 4) and NOD/ Scid ( n = 4) mice (6-10 weeks of age) received a total of six subcutaneous (s.c.) injections of 1 × 10 4 ( n = 2), 1 × 10 5 ( n = 2), and 1 × 10 6 ( n = 2) midpassage MSC- Luc2 cells/mouse. Untreated age-matched NOD ( n = 2) and NOD/ Scid ( n = 2) mice were utilized as negative controls. BLI images were acquired following i.p. administration of D-luciferin (15 mg/ml) at 150 mg/kg or 10 μ l/g. Images are representative of a single experimental NOD/ Scid and NOD animal. (b) Analysis of MSC BLI in NOD and NOD/ Scid mice. Regions of interest were established surrounding the areas corresponding to the sites of cell transplantation using the Living Image 3.1 (PerkinElmer™) software. Quantitative data were subsequently analyzed using GraphPad Prism 7 ® . Data were presented as mean radiance ± SEM over time (weeks). A two-way ANOVA and Tukey's posttests were performed, ∗ p
    Figure Legend Snippet: Persistence of syngeneic MSCs in immune-competent and immune-deficient animal models. (a) NOD ( n = 4) and NOD/ Scid ( n = 4) mice (6-10 weeks of age) received a total of six subcutaneous (s.c.) injections of 1 × 10 4 ( n = 2), 1 × 10 5 ( n = 2), and 1 × 10 6 ( n = 2) midpassage MSC- Luc2 cells/mouse. Untreated age-matched NOD ( n = 2) and NOD/ Scid ( n = 2) mice were utilized as negative controls. BLI images were acquired following i.p. administration of D-luciferin (15 mg/ml) at 150 mg/kg or 10 μ l/g. Images are representative of a single experimental NOD/ Scid and NOD animal. (b) Analysis of MSC BLI in NOD and NOD/ Scid mice. Regions of interest were established surrounding the areas corresponding to the sites of cell transplantation using the Living Image 3.1 (PerkinElmer™) software. Quantitative data were subsequently analyzed using GraphPad Prism 7 ® . Data were presented as mean radiance ± SEM over time (weeks). A two-way ANOVA and Tukey's posttests were performed, ∗ p

    Techniques Used: Mouse Assay, Transplantation Assay, Software

    NOD-derived MSC nucleofection. (a) MSCs (early passage number) were nucleofected with 0 and 5 μ g pVITRO2- Luc2 . Parental MSCs and MSC- Luc2 at an equivalent passage number (P15) showed native fibroblast-like morphology and maintained plastic adherence and self-renewal properties. Images were acquired on a Leica DM light microscope at 10x magnification, scale bar = 100 μ m. (b) In vitro functional characterization of luciferase activity in MSC- Luc2. Cells were incubated with 1 : 1 D-luciferin (300 μ g/ml) and imaged on the IVIS Lumina II, according to the in vitro BLI acquisition settings. The image represented is at t = 30 min after the addition of D-luciferin. Lane 1: D-PBS; lanes 2-10: MSC- Luc2 and MSC- Luc2/LacZ (control). (c) Linear regression analysis of luminescent signal was performed using GraphPad Prism 7 ® . Data are presented as means ± SDs of triplicates.
    Figure Legend Snippet: NOD-derived MSC nucleofection. (a) MSCs (early passage number) were nucleofected with 0 and 5 μ g pVITRO2- Luc2 . Parental MSCs and MSC- Luc2 at an equivalent passage number (P15) showed native fibroblast-like morphology and maintained plastic adherence and self-renewal properties. Images were acquired on a Leica DM light microscope at 10x magnification, scale bar = 100 μ m. (b) In vitro functional characterization of luciferase activity in MSC- Luc2. Cells were incubated with 1 : 1 D-luciferin (300 μ g/ml) and imaged on the IVIS Lumina II, according to the in vitro BLI acquisition settings. The image represented is at t = 30 min after the addition of D-luciferin. Lane 1: D-PBS; lanes 2-10: MSC- Luc2 and MSC- Luc2/LacZ (control). (c) Linear regression analysis of luminescent signal was performed using GraphPad Prism 7 ® . Data are presented as means ± SDs of triplicates.

    Techniques Used: Derivative Assay, Light Microscopy, In Vitro, Functional Assay, Luciferase, Activity Assay, Incubation

    11) Product Images from "Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major)"

    Article Title: Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major)

    Journal: Archives of toxicology

    doi: 10.1007/s00204-016-1732-9

    Schematic diagram of rsAHR1/2 direct interaction with XREs in 5′-flanking region of rsAHR2 gene. TCDD-activated rsAHR1 and rsAHR2 bind to XRE site and induce rsAHR2 gene expression. rsAHR1 specific interaction is shown in XRE2.
    Figure Legend Snippet: Schematic diagram of rsAHR1/2 direct interaction with XREs in 5′-flanking region of rsAHR2 gene. TCDD-activated rsAHR1 and rsAHR2 bind to XRE site and induce rsAHR2 gene expression. rsAHR1 specific interaction is shown in XRE2.

    Techniques Used: Expressing

    Nucleotide sequences of 5′ -flanking region of the rsAHR1 (a) and rsAHR2 (b) genes. Schematic diagram of the 5′-flanking region of each AHR gene is shown at the top. The XRE sequence indicated as a solid box is shown at the 5′-flanking region from the putative transcription start site. Nucleotides are numbered with negative numbers representing the 5′-flanking region from the putative transcription start site (ATG). The transcription start site and translation start site are marked by arrows. The putative XRE-like sites identified using MatInspector and TRANSFAC are boxed. Other pertinent sequences are labeled and indicated by underline. CAAT box (the binding site for the RNA transcription factor); GC boxes (the binding sites for SP1); activator protein-1 (AP-1) binding site; RELA (NF-κB binding site); RARE (retinoic acid binding site); CRE (the cAMP binding site)
    Figure Legend Snippet: Nucleotide sequences of 5′ -flanking region of the rsAHR1 (a) and rsAHR2 (b) genes. Schematic diagram of the 5′-flanking region of each AHR gene is shown at the top. The XRE sequence indicated as a solid box is shown at the 5′-flanking region from the putative transcription start site. Nucleotides are numbered with negative numbers representing the 5′-flanking region from the putative transcription start site (ATG). The transcription start site and translation start site are marked by arrows. The putative XRE-like sites identified using MatInspector and TRANSFAC are boxed. Other pertinent sequences are labeled and indicated by underline. CAAT box (the binding site for the RNA transcription factor); GC boxes (the binding sites for SP1); activator protein-1 (AP-1) binding site; RELA (NF-κB binding site); RARE (retinoic acid binding site); CRE (the cAMP binding site)

    Techniques Used: Sequencing, Labeling, Binding Assay

    Transactivation potencies of the 5′-flanking region of rsAHR2 gene by rsAHR1 (a) and rsAHR2 (b) treated with TCDD. Values represent the mean ± SD. The concentration-response curves were derived from at least 6 replicates from 2 independent experiments for each concentration.
    Figure Legend Snippet: Transactivation potencies of the 5′-flanking region of rsAHR2 gene by rsAHR1 (a) and rsAHR2 (b) treated with TCDD. Values represent the mean ± SD. The concentration-response curves were derived from at least 6 replicates from 2 independent experiments for each concentration.

    Techniques Used: Concentration Assay, Derivative Assay

    Results of XRE point mutation assay of rsAHR2 gene by rsAHR1 and rsAHR2 treated with TCDD. Schematic diagrams of wild type and point mutated XREs in 5′-flanking region of rsAHR2 gene (a). Transactivation potencies of rsAHR2 gene by rsAHR1 (b) and rsAHR2 (c) treated with TCDD. Different letters on each bar denote significant differences between DMSO- and TCDD-treated cells ( p
    Figure Legend Snippet: Results of XRE point mutation assay of rsAHR2 gene by rsAHR1 and rsAHR2 treated with TCDD. Schematic diagrams of wild type and point mutated XREs in 5′-flanking region of rsAHR2 gene (a). Transactivation potencies of rsAHR2 gene by rsAHR1 (b) and rsAHR2 (c) treated with TCDD. Different letters on each bar denote significant differences between DMSO- and TCDD-treated cells ( p

    Techniques Used: Point Mutation Assay

    12) Product Images from "Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands"

    Article Title: Detection of the Rhoptry Neck Protein Complex in Plasmodium Sporozoites and Its Contribution to Sporozoite Invasion of Salivary Glands

    Journal: mSphere

    doi: 10.1128/mSphere.00325-20

    Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P
    Figure Legend Snippet: Generation of sporozoite-stage-specific ron4 or ron5 knockdown transgenic parasites. (A) Genomic Southern blot analyses using genomic DNA extracted from Pb WT-GFP and transgenic parasites. Genomic DNA was digested with BamHI and EcoRV, separated by size via agarose gel electrophoresis, and transferred to membranes. The signals were obtained by hybridization of the DNA probe within the hDHFR cassette at expected sizes indicated by closed and open arrowheads for conditional knockdown and control parasites, respectively. The specific bands at the expected size (6.4, 5.9, 5.7, and 5.5 kbp for RON4-cKD, RON4-cont, RON5-cKD, and RON5-cont, respectively) (see Fig. S3A and B ) demonstrate that DNA insertion occurred at the correct locus to replace the original promoter regions. The DNA size marker is shown on the left of each image. (B) Relative amounts of ron4 and ron5 mRNA in oocyst-derived sporozoites. Total RNA was extracted from mosquito midguts collected at days 14 to 15 postfeeding that were infected with RON4-cont, RON4-cKD cl1, RON4-cKD cl2, RON5-cont, RON5-cKD cl1, and RON5-cKD cl2. The values were normalized by the expression of pbhsp70 mRNA in each sample. Experiments were repeated at least four times with independently prepared sets of samples (see Fig. S3C ), and the means with standard deviations are shown as bar graphs. Statistical differences were calculated by one-way ANOVA with Tukey’s multiple-comparison test (****, P

    Techniques Used: Transgenic Assay, Southern Blot, Agarose Gel Electrophoresis, Hybridization, Marker, Derivative Assay, Infection, Expressing

    13) Product Images from "GPR41 Gene Expression Is Mediated by Internal Ribosome Entry Site (IRES)-dependent Translation of Bicistronic mRNA Encoding GPR40 and GPR41 Proteins *"

    Article Title: GPR41 Gene Expression Is Mediated by Internal Ribosome Entry Site (IRES)-dependent Translation of Bicistronic mRNA Encoding GPR40 and GPR41 Proteins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.358887

    Genome structure and gene expression pattern of GPR40/41 locus. A , representation of the GPR40/41 locus. GPR40 and GPR41 ORFs are shown as black boxes. White boxes flanking the GPR40 ORF indicate 5′- and 3′UTRs. Boxes upstream of the GPR40
    Figure Legend Snippet: Genome structure and gene expression pattern of GPR40/41 locus. A , representation of the GPR40/41 locus. GPR40 and GPR41 ORFs are shown as black boxes. White boxes flanking the GPR40 ORF indicate 5′- and 3′UTRs. Boxes upstream of the GPR40

    Techniques Used: Expressing

    Validation of bicistronic mRNA expression. A , illustration of siRNA system. The small bars represent targeted areas of GPR40/41. Each pool consists of four different siRNAs targeting the coding region of GPR40 or GPR41 as indicated. B , knockdown of GPR40
    Figure Legend Snippet: Validation of bicistronic mRNA expression. A , illustration of siRNA system. The small bars represent targeted areas of GPR40/41. Each pool consists of four different siRNAs targeting the coding region of GPR40 or GPR41 as indicated. B , knockdown of GPR40

    Techniques Used: Expressing

    Analysis of transcriptional and translational control of GPR40 and GPR41 gene expression using bicistronic luciferase constructs. A , bicistronic vectors used in this analysis. WT represents the genomic organization of the locus; open reading frames are
    Figure Legend Snippet: Analysis of transcriptional and translational control of GPR40 and GPR41 gene expression using bicistronic luciferase constructs. A , bicistronic vectors used in this analysis. WT represents the genomic organization of the locus; open reading frames are

    Techniques Used: Expressing, Luciferase, Construct

    Analysis of GPR41 gene expression by 5′-RACE and reporter gene assay. A , map illustrating GPR40/41 locus. GPR40 and GPR41 coding regions are shown as black boxes. White boxes indicate 5′- and 3′UTRs of the GPR40 gene and 3′UTR
    Figure Legend Snippet: Analysis of GPR41 gene expression by 5′-RACE and reporter gene assay. A , map illustrating GPR40/41 locus. GPR40 and GPR41 coding regions are shown as black boxes. White boxes indicate 5′- and 3′UTRs of the GPR40 gene and 3′UTR

    Techniques Used: Expressing, Reporter Gene Assay

    Analysis of GPR40 and GPR41 transcripts by Northern blot and 5′-RACE. A, probes a–d correspond to GPR40 ORF, GPR40 3′UTR, intergenic region, and GPR41 ORF, respectively. The location of primers used for 5′-RACE is indicated
    Figure Legend Snippet: Analysis of GPR40 and GPR41 transcripts by Northern blot and 5′-RACE. A, probes a–d correspond to GPR40 ORF, GPR40 3′UTR, intergenic region, and GPR41 ORF, respectively. The location of primers used for 5′-RACE is indicated

    Techniques Used: Northern Blot

    14) Product Images from "Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿"

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01135-10

    Restriction digest patterns electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. (A) FnpΦ02 genomic DNA digested with restriction enzymes. Lanes: L1, 100-bp DNA ladder marker (Fermentas Inc., Canada); L2, 1-kb ladder marker (Fermentas Inc., Canada); L3, undigested DNA; L4, DNA/HindIII; L5, DNA/DraI; L6, DNA/XbaI; and L7, lambda DNA/HindIII. (B) Higher magnification of the bands of the digestion of FnpΦ02 with HindIII used for genome size determination. Selected sizes of the marker are indicated in panel A.
    Figure Legend Snippet: Restriction digest patterns electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. (A) FnpΦ02 genomic DNA digested with restriction enzymes. Lanes: L1, 100-bp DNA ladder marker (Fermentas Inc., Canada); L2, 1-kb ladder marker (Fermentas Inc., Canada); L3, undigested DNA; L4, DNA/HindIII; L5, DNA/DraI; L6, DNA/XbaI; and L7, lambda DNA/HindIII. (B) Higher magnification of the bands of the digestion of FnpΦ02 with HindIII used for genome size determination. Selected sizes of the marker are indicated in panel A.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Marker, Lambda DNA Preparation

    Related Articles

    Amplification:

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    Article Snippet: .. Genomic DNA was extracted from the cell line using the Wizard SV genomic DNA purification system (Promega), and amplified by PCR using the primers designed to cover the entire socs-3 gene on chromosome 17q25.3 (GI: 37542591) consisting of 2 exons, 1 intron and 5′- and 3′-franking regions ( ). .. PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, visualized under ultraviolet illumination and purified using a gel extraction kit (Qiagen, Valencia, CA).

    Methylation:

    Article Title: Regulation of ALF Promoter Activity in Xenopus Oocytes
    Article Snippet: .. Bisulfite methylation analysis Genomic DNA was isolated from Xenopus laevis oocytes and liver using the Wizard SV Genomic DNA Purification System (Promega) and processed using the EpiTect Bisulfite Kit (Qiagen). .. In brief, DNAs were subject to repeated denaturing (99°C for 5 min) with several incubation steps (60°C for 25 min, 85 min, and 175 min).

    Isolation:

    Article Title: Regulation of ALF Promoter Activity in Xenopus Oocytes
    Article Snippet: .. Bisulfite methylation analysis Genomic DNA was isolated from Xenopus laevis oocytes and liver using the Wizard SV Genomic DNA Purification System (Promega) and processed using the EpiTect Bisulfite Kit (Qiagen). .. In brief, DNAs were subject to repeated denaturing (99°C for 5 min) with several incubation steps (60°C for 25 min, 85 min, and 175 min).

    Article Title: Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids
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    Cell Culture:

    Article Title: Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction
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    Purification:

    Article Title: Immortalization of CD4+ and CD8+ T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
    Article Snippet: .. Genomic DNA was purified from ACH.pcTax- and ACH.M47-immortalized cell lines by the Wizard Prep genomic DNA purification system (Promega, Madison, Wis.) according to the manufacturer’s instructions. .. PCR (95°C for 1 min; 60°C for 2 min; 72°C for 3 min; 35 cycles) was performed on 200 ng of genomic DNA with the following primers: 5′-CGGAATTCATGGCCCACTTCCCAGGGTTTGG-3′ and 5′-CGGGATCCCTAGTCACTTAGACTTCTGTTTCTCGGAAATG-3′, which amplifies the entire Tax open reading frame (ORF).

    Article Title: Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum
    Article Snippet: .. DNA fragments were precipitated with absolute ethanol, dried at room temperature, dissolved in 20 μL of distilled water, re-amplified using the corresponding RAPD or AFLP primers and purified with the DNA Wizard SV Gel and PCR Clean-up system (Promega). .. Clean fragments were cloned with the pGEM-T Easy Vector system (Promega).

    Article Title: The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells
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    Real-time Polymerase Chain Reaction:

    Article Title: The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells
    Article Snippet: .. PCR and Real-time PCR Total cellular DNA was purified using the Wizard SV Genomic DNA Purification System as recommended by the manufacturer (Promega). .. The detection of viral late DNA and 1-LTR-circles by PCR was performed as described previously .

    DNA Purification:

    Article Title: Regulation of ALF Promoter Activity in Xenopus Oocytes
    Article Snippet: .. Bisulfite methylation analysis Genomic DNA was isolated from Xenopus laevis oocytes and liver using the Wizard SV Genomic DNA Purification System (Promega) and processed using the EpiTect Bisulfite Kit (Qiagen). .. In brief, DNAs were subject to repeated denaturing (99°C for 5 min) with several incubation steps (60°C for 25 min, 85 min, and 175 min).

    Article Title: Immortalization of CD4+ and CD8+ T Lymphocytes by Human T-Cell Leukemia Virus Type 1 Tax Mutants Expressed in a Functional Molecular Clone
    Article Snippet: .. Genomic DNA was purified from ACH.pcTax- and ACH.M47-immortalized cell lines by the Wizard Prep genomic DNA purification system (Promega, Madison, Wis.) according to the manufacturer’s instructions. .. PCR (95°C for 1 min; 60°C for 2 min; 72°C for 3 min; 35 cycles) was performed on 200 ng of genomic DNA with the following primers: 5′-CGGAATTCATGGCCCACTTCCCAGGGTTTGG-3′ and 5′-CGGGATCCCTAGTCACTTAGACTTCTGTTTCTCGGAAATG-3′, which amplifies the entire Tax open reading frame (ORF).

    Article Title: The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells
    Article Snippet: .. PCR and Real-time PCR Total cellular DNA was purified using the Wizard SV Genomic DNA Purification System as recommended by the manufacturer (Promega). .. The detection of viral late DNA and 1-LTR-circles by PCR was performed as described previously .

    Article Title: Sustained IL-6/STAT-3 Signaling in Cholangiocarcinoma Cells due to SOCS-3 Epigenetic Silencing
    Article Snippet: .. Genomic DNA was extracted from the cell line using the Wizard SV genomic DNA purification system (Promega), and amplified by PCR using the primers designed to cover the entire socs-3 gene on chromosome 17q25.3 (GI: 37542591) consisting of 2 exons, 1 intron and 5′- and 3′-franking regions ( ). .. PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, visualized under ultraviolet illumination and purified using a gel extraction kit (Qiagen, Valencia, CA).

    Article Title: Targeted disruption of the CCR5 gene in human hematopoietic stem cells stimulated by peptide nucleic acids
    Article Snippet: .. Genomic DNA was isolated from tissue culture samples using the Wizard SV Genomic DNA Purification System (Promega); from THP-1 cells in 96-well plates, the Wizard SV 96 Genomic DNA Purification System was used. .. RNA was isolated using the Absolutely RNA Miniprep Kit (Stratagene).

    Article Title: Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction
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    Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
    Article Snippet: .. Unless otherwise stated, pUC18 form I DNA was prepared from E.coli JM109 with the Promega Wizard Plus DNA purification system followed by ethanol precipitation. .. Alternatively, pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange and gel filtration chromatography [Q high (BioRad) and Chroma Spin + TE-1000 columns (BD Biosciences)].

    Ethanol Precipitation:

    Article Title: On the mechanism of strand assimilation by the herpes simplex virus type-1 single-strand DNA-binding protein (ICP8)
    Article Snippet: .. Unless otherwise stated, pUC18 form I DNA was prepared from E.coli JM109 with the Promega Wizard Plus DNA purification system followed by ethanol precipitation. .. Alternatively, pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange and gel filtration chromatography [Q high (BioRad) and Chroma Spin + TE-1000 columns (BD Biosciences)].

    Polymerase Chain Reaction:

    Article Title: Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum
    Article Snippet: .. DNA fragments were precipitated with absolute ethanol, dried at room temperature, dissolved in 20 μL of distilled water, re-amplified using the corresponding RAPD or AFLP primers and purified with the DNA Wizard SV Gel and PCR Clean-up system (Promega). .. Clean fragments were cloned with the pGEM-T Easy Vector system (Promega).

    Article Title: The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells
    Article Snippet: .. PCR and Real-time PCR Total cellular DNA was purified using the Wizard SV Genomic DNA Purification System as recommended by the manufacturer (Promega). .. The detection of viral late DNA and 1-LTR-circles by PCR was performed as described previously .

    Article Title: Sustained IL-6/STAT-3 Signaling in Cholangiocarcinoma Cells due to SOCS-3 Epigenetic Silencing
    Article Snippet: .. Genomic DNA was extracted from the cell line using the Wizard SV genomic DNA purification system (Promega), and amplified by PCR using the primers designed to cover the entire socs-3 gene on chromosome 17q25.3 (GI: 37542591) consisting of 2 exons, 1 intron and 5′- and 3′-franking regions ( ). .. PCR products were electrophoresed on a 1% agarose gel, stained with ethidium bromide, visualized under ultraviolet illumination and purified using a gel extraction kit (Qiagen, Valencia, CA).

    TaqMan SNP Genotyping Assay:

    Article Title: Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction
    Article Snippet: .. Determination of genotypes Genomic DNA was extracted from cultured SMCs and ECs or from sections of formaldehyde-fixed paraffin-embedded blocks of atherosclerotic coronary arteries using the Wizard SV Genomic DNA Purification System (Promega). rs4773144 genotypes were determined with the use of the TaqMan SNP genotyping assay. .. Accuracy of the genotyping results was verified by sequencing of a random selection of the samples.

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