expand high fidelity plus pcr system  (Roche)


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    Structured Review

    Roche expand high fidelity plus pcr system
    <t>Phox2b</t> deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real <t>time-PCR</t> analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P
    Expand High Fidelity Plus Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity plus pcr system/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity plus pcr system - by Bioz Stars, 2021-09
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    Images

    1) Product Images from "Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models"

    Article Title: Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003533

    Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P
    Figure Legend Snippet: Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P

    Techniques Used: In Situ Hybridization, Labeling, Expressing, Injection, Real-time Polymerase Chain Reaction

    2) Product Images from "The lymphotoxin LT?1?2 controls postnatal and adult spleen marginal sinus vascular structure and function"

    Article Title: The lymphotoxin LT?1?2 controls postnatal and adult spleen marginal sinus vascular structure and function

    Journal: Immunity

    doi: 10.1016/j.immuni.2009.01.010

    Endothelial cells express the LTβR and utilize MAdCAM-1 to form specialized structures on Matrigel (A) Ten µm spleen sections from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n=3). Endothelial cells isolated from WT spleen (sECs) using either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n=3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR. (F) sECs isolated from WT and Lta −/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days. (G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 µg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hrs. (H) Protein extracts from transfected bEND.3 cells were prepared and an ELISA was performed to assess MAdCAM-1 protein expression. (I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T. and 8 µm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue). Inset images show magnified areas highlighting the EC phenotype and amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of 3 independent experiments.
    Figure Legend Snippet: Endothelial cells express the LTβR and utilize MAdCAM-1 to form specialized structures on Matrigel (A) Ten µm spleen sections from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n=3). Endothelial cells isolated from WT spleen (sECs) using either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n=3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR. (F) sECs isolated from WT and Lta −/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days. (G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 µg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hrs. (H) Protein extracts from transfected bEND.3 cells were prepared and an ELISA was performed to assess MAdCAM-1 protein expression. (I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T. and 8 µm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue). Inset images show magnified areas highlighting the EC phenotype and amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of 3 independent experiments.

    Techniques Used: Mouse Assay, Staining, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Characterization of arrangement and expression of the T cell receptor ? locus in the sandbar shark"

    Article Title: Characterization of arrangement and expression of the T cell receptor ? locus in the sandbar shark

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

    doi: 10.1073/pnas.0811283106

    Southern blottings of the shark TCR γ locus. Sandbar shark spleen DNA was digested by BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) independently. The probe was full-length C region isolated by PCR of cDNA.
    Figure Legend Snippet: Southern blottings of the shark TCR γ locus. Sandbar shark spleen DNA was digested by BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) independently. The probe was full-length C region isolated by PCR of cDNA.

    Techniques Used: Isolation, Polymerase Chain Reaction

    Related Articles

    Plasmid Preparation:

    Article Title: Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes
    Article Snippet: .. Plasmid construction and generation of transgenic tomato roots All PCR amplification used for plasmids construction were performed using the Expand High Fidelity plus PCR System (Roche) according to the manufacturer's instructions. ..

    Transgenic Assay:

    Article Title: Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes
    Article Snippet: .. Plasmid construction and generation of transgenic tomato roots All PCR amplification used for plasmids construction were performed using the Expand High Fidelity plus PCR System (Roche) according to the manufacturer's instructions. ..

    Polymerase Chain Reaction:

    Article Title: Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes
    Article Snippet: .. Plasmid construction and generation of transgenic tomato roots All PCR amplification used for plasmids construction were performed using the Expand High Fidelity plus PCR System (Roche) according to the manufacturer's instructions. ..

    Article Title: Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models
    Article Snippet: .. 50 pg of 1st strand cDNA was used to amplify phox2b using the Expand High Fidelity Plus PCR system (Roche). ..

    Article Title: Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †
    Article Snippet: .. Mutations that prematurely terminated Rev translation at an arginine residue at amino acid 38 (R38), removed the Rev AUG codon (Rev− ) and/or the Vpu AUG codon (Vpu− ), increased the Kozak strength of the Rev AUG codon, added a strong Kozak AUG codon to exon 2, or mutated this AUG codon to AGG were all synthesized by PCR mutagenesis by using the Expand high-fidelity PCR system (Roche) and the following protocol with the template and primer sets outlined in Table . ..

    Article Title: A novel eukaryotic factor for cytosolic Fe-S cluster assembly
    Article Snippet: .. To clone the CFD1 gene, yeast genomic DNA was prepared as previously described ( ) and used for amplification of CFD1 genes by the polymerase chain reaction (PCR) using the Expand High Fidelity PCR System (Roche), according to the manufacturer’s recommendation. ..

    Article Title: The lymphotoxin LT?1?2 controls postnatal and adult spleen marginal sinus vascular structure and function
    Article Snippet: .. Murine MAdCAM-1 cDNA corresponding to nt 21 to 1243 ( ) was amplified from bEND.3 mRNA with the Expand High Fidelity Plus PCR system (Roche). ..

    Article Title: Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila
    Article Snippet: .. Molecular Biology For the Actin88F:eGFP construct, a 2053 bp region immediately upstream of the actin88F gene was amplified by the Expand High Fidelity PLUS PCR system (Roche) from Oregon-R genomic DNA using the following forward primer containing a BmtI site: 5′-GCT AGC ATG CAC AAT AGG CAA ATT TAG TT-3′ and reverse primer containing an EcoRI site: 5′-GAA TTC CTT GGC AGT TGT TTA TCT GGA A-3′. eGFP was similarly amplified using the following forward primer containing a KpnI site: 5′-GGT ACC ATG GTG AGC AAG GGC GA-3′ and reverse primer containing an XbaI site: 5′-TCT AGA TTA CTT GTA CAG CTC GTC CAT GC-3′. ..

    Article Title: Characterization of arrangement and expression of the T cell receptor ? locus in the sandbar shark
    Article Snippet: .. Multiple DNA polymerase kits, including Expand High Fidelity Plus PCR system (Roche), Expand 20 kb Plus PCR system (Roche), and iProof High Fidelity DNA polymerase (Bio-Rad), were used to eliminate possible PCR errors and to increase the chances of getting the right DNA fragment. ..

    Amplification:

    Article Title: Fatty Acid-and Retinol-Binding Protein, Mj-FAR-1 Induces Tomato Host Susceptibility to Root-Knot Nematodes
    Article Snippet: .. Plasmid construction and generation of transgenic tomato roots All PCR amplification used for plasmids construction were performed using the Expand High Fidelity plus PCR System (Roche) according to the manufacturer's instructions. ..

    Article Title: A novel eukaryotic factor for cytosolic Fe-S cluster assembly
    Article Snippet: .. To clone the CFD1 gene, yeast genomic DNA was prepared as previously described ( ) and used for amplification of CFD1 genes by the polymerase chain reaction (PCR) using the Expand High Fidelity PCR System (Roche), according to the manufacturer’s recommendation. ..

    Article Title: The lymphotoxin LT?1?2 controls postnatal and adult spleen marginal sinus vascular structure and function
    Article Snippet: .. Murine MAdCAM-1 cDNA corresponding to nt 21 to 1243 ( ) was amplified from bEND.3 mRNA with the Expand High Fidelity Plus PCR system (Roche). ..

    Article Title: Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila
    Article Snippet: .. Molecular Biology For the Actin88F:eGFP construct, a 2053 bp region immediately upstream of the actin88F gene was amplified by the Expand High Fidelity PLUS PCR system (Roche) from Oregon-R genomic DNA using the following forward primer containing a BmtI site: 5′-GCT AGC ATG CAC AAT AGG CAA ATT TAG TT-3′ and reverse primer containing an EcoRI site: 5′-GAA TTC CTT GGC AGT TGT TTA TCT GGA A-3′. eGFP was similarly amplified using the following forward primer containing a KpnI site: 5′-GGT ACC ATG GTG AGC AAG GGC GA-3′ and reverse primer containing an XbaI site: 5′-TCT AGA TTA CTT GTA CAG CTC GTC CAT GC-3′. ..

    Synthesized:

    Article Title: Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †
    Article Snippet: .. Mutations that prematurely terminated Rev translation at an arginine residue at amino acid 38 (R38), removed the Rev AUG codon (Rev− ) and/or the Vpu AUG codon (Vpu− ), increased the Kozak strength of the Rev AUG codon, added a strong Kozak AUG codon to exon 2, or mutated this AUG codon to AGG were all synthesized by PCR mutagenesis by using the Expand high-fidelity PCR system (Roche) and the following protocol with the template and primer sets outlined in Table . ..

    Mutagenesis:

    Article Title: Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †
    Article Snippet: .. Mutations that prematurely terminated Rev translation at an arginine residue at amino acid 38 (R38), removed the Rev AUG codon (Rev− ) and/or the Vpu AUG codon (Vpu− ), increased the Kozak strength of the Rev AUG codon, added a strong Kozak AUG codon to exon 2, or mutated this AUG codon to AGG were all synthesized by PCR mutagenesis by using the Expand high-fidelity PCR system (Roche) and the following protocol with the template and primer sets outlined in Table . ..

    Construct:

    Article Title: Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila
    Article Snippet: .. Molecular Biology For the Actin88F:eGFP construct, a 2053 bp region immediately upstream of the actin88F gene was amplified by the Expand High Fidelity PLUS PCR system (Roche) from Oregon-R genomic DNA using the following forward primer containing a BmtI site: 5′-GCT AGC ATG CAC AAT AGG CAA ATT TAG TT-3′ and reverse primer containing an EcoRI site: 5′-GAA TTC CTT GGC AGT TGT TTA TCT GGA A-3′. eGFP was similarly amplified using the following forward primer containing a KpnI site: 5′-GGT ACC ATG GTG AGC AAG GGC GA-3′ and reverse primer containing an XbaI site: 5′-TCT AGA TTA CTT GTA CAG CTC GTC CAT GC-3′. ..

    Cellular Antioxidant Activity Assay:

    Article Title: Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila
    Article Snippet: .. Molecular Biology For the Actin88F:eGFP construct, a 2053 bp region immediately upstream of the actin88F gene was amplified by the Expand High Fidelity PLUS PCR system (Roche) from Oregon-R genomic DNA using the following forward primer containing a BmtI site: 5′-GCT AGC ATG CAC AAT AGG CAA ATT TAG TT-3′ and reverse primer containing an EcoRI site: 5′-GAA TTC CTT GGC AGT TGT TTA TCT GGA A-3′. eGFP was similarly amplified using the following forward primer containing a KpnI site: 5′-GGT ACC ATG GTG AGC AAG GGC GA-3′ and reverse primer containing an XbaI site: 5′-TCT AGA TTA CTT GTA CAG CTC GTC CAT GC-3′. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Fluorescence Behavioral Imaging (FBI) Tracks Identity in Heterogeneous Groups of Drosophila
    Article Snippet: .. Molecular Biology For the Actin88F:eGFP construct, a 2053 bp region immediately upstream of the actin88F gene was amplified by the Expand High Fidelity PLUS PCR system (Roche) from Oregon-R genomic DNA using the following forward primer containing a BmtI site: 5′-GCT AGC ATG CAC AAT AGG CAA ATT TAG TT-3′ and reverse primer containing an EcoRI site: 5′-GAA TTC CTT GGC AGT TGT TTA TCT GGA A-3′. eGFP was similarly amplified using the following forward primer containing a KpnI site: 5′-GGT ACC ATG GTG AGC AAG GGC GA-3′ and reverse primer containing an XbaI site: 5′-TCT AGA TTA CTT GTA CAG CTC GTC CAT GC-3′. ..

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    Roche expand high fidelity plus pcr system
    <t>Phox2b</t> deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real <t>time-PCR</t> analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P
    Expand High Fidelity Plus Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity plus pcr system/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity plus pcr system - by Bioz Stars, 2021-09
    86/100 stars
      Buy from Supplier

    86
    Roche dna polymerase
    AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro . A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a <t>DNA/protein</t> complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. <t>RT-PCR</t> amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.
    Dna Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2021-09
    86/100 stars
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    Image Search Results


    Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P

    Journal: PLoS Genetics

    Article Title: Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

    doi: 10.1371/journal.pgen.1003533

    Figure Lengend Snippet: Phox2b deficiency causes arrest of SCG cells at an undifferentiated stage. (A,B) Dorsal views of ISH with FITC-labeled th (red) and digoxigenin-labeled phox2b (blue) riboprobes on 4-dpf embryos in which phox2b expression was abrogated by MO knockdown. Arrows point to the SCG. Insets show enlarged views of the SCG. (C, D) Lateral views of ISH with FITC-labeled phox2b (red) and digoxigenin-labeled dbh (blue) riboprobes in MO-injected (D) and control (C) embryos. (E) Quantitative real time-PCR analysis comparing phox2b mRNA expression levels in control vs. phox2b MO-injected (P2BATG MO) embryos. Data are presented as means ± SD ( **** P

    Article Snippet: 50 pg of 1st strand cDNA was used to amplify phox2b using the Expand High Fidelity Plus PCR system (Roche).

    Techniques: In Situ Hybridization, Labeling, Expressing, Injection, Real-time Polymerase Chain Reaction

    Endothelial cells express the LTβR and utilize MAdCAM-1 to form specialized structures on Matrigel (A) Ten µm spleen sections from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n=3). Endothelial cells isolated from WT spleen (sECs) using either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n=3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR. (F) sECs isolated from WT and Lta −/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days. (G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 µg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hrs. (H) Protein extracts from transfected bEND.3 cells were prepared and an ELISA was performed to assess MAdCAM-1 protein expression. (I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T. and 8 µm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue). Inset images show magnified areas highlighting the EC phenotype and amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of 3 independent experiments.

    Journal: Immunity

    Article Title: The lymphotoxin LT?1?2 controls postnatal and adult spleen marginal sinus vascular structure and function

    doi: 10.1016/j.immuni.2009.01.010

    Figure Lengend Snippet: Endothelial cells express the LTβR and utilize MAdCAM-1 to form specialized structures on Matrigel (A) Ten µm spleen sections from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n=3). Endothelial cells isolated from WT spleen (sECs) using either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n=3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR. (F) sECs isolated from WT and Lta −/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days. (G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 µg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hrs. (H) Protein extracts from transfected bEND.3 cells were prepared and an ELISA was performed to assess MAdCAM-1 protein expression. (I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T. and 8 µm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue). Inset images show magnified areas highlighting the EC phenotype and amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of 3 independent experiments.

    Article Snippet: Murine MAdCAM-1 cDNA corresponding to nt 21 to 1243 ( ) was amplified from bEND.3 mRNA with the Expand High Fidelity Plus PCR system (Roche).

    Techniques: Mouse Assay, Staining, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    Southern blottings of the shark TCR γ locus. Sandbar shark spleen DNA was digested by BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) independently. The probe was full-length C region isolated by PCR of cDNA.

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

    Article Title: Characterization of arrangement and expression of the T cell receptor ? locus in the sandbar shark

    doi: 10.1073/pnas.0811283106

    Figure Lengend Snippet: Southern blottings of the shark TCR γ locus. Sandbar shark spleen DNA was digested by BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) independently. The probe was full-length C region isolated by PCR of cDNA.

    Article Snippet: Multiple DNA polymerase kits, including Expand High Fidelity Plus PCR system (Roche), Expand 20 kb Plus PCR system (Roche), and iProof High Fidelity DNA polymerase (Bio-Rad), were used to eliminate possible PCR errors and to increase the chances of getting the right DNA fragment.

    Techniques: Isolation, Polymerase Chain Reaction

    AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro . A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a DNA/protein complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. RT-PCR amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.

    Journal: PLoS ONE

    Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts

    doi: 10.1371/journal.pone.0066892

    Figure Lengend Snippet: AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro . A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a DNA/protein complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. RT-PCR amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.

    Article Snippet: PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes.

    Techniques: In Vivo, In Vitro, Electrophoretic Mobility Shift Assay, Protein Binding, Incubation, Sequencing, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Amplification