cdna Thermo Fisher Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher complementary dna cdna synthesis kit
    Among VLP constituents, Vpx is necessary and sufficient to rescue HIV-1 from type I IFN . ( A ) MDDCs were treated with LPS for 24 hrs, then treated for 3 hrs with media or the indicated VSV-G-pseudotyped HIV-2 ROD or SIV MAC-251 VLPs, and finally challenged with a VSV-G-pseudotyped HIV-1 NL4-3 GFP reporter virus. Infectivity was measured by flow cytometry. ( B ) As indicated, 293T cells were co-transfected with a codon optimized SIV MAC251 vpx expression plasmid and HIV-1 GFP reporter vectors bearing either wild-type Gag or Gag with an engineered Vpx binding motif (DPAVDLL). Proteins from the cell lysate and from virion preparations were separated by SDS-PAGE and then immunoblotted with anti-Vpx or anti-p24 antibodies. ( C ) MDDCs treated with IFN-β for 24 h and were then challenged with VSV-G-pseudotyped HIV-1 GFP reporter vectors with wild-type HIV-1 Gag or HIV-1 Gag bearing the engineered Vpx binding motif (DPAVDLL). Both HIV-1 reporter vectors were produced in the presence of empty pcDNA3.1 plasmid or pcDNA3.1 containing a codon-optimized SIV MAC-251 vpx <t>cDNA.</t> Data are representative of one of at least three independent experiments. Error bars represent ± SD (n = 3).
    Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1049 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna synthesis kit/product/Thermo Fisher
    Average 99 stars, based on 1049 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna synthesis kit - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher complementary dna cdna
    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary <t>DNA</t> was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22625 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna/product/Thermo Fisher
    Average 99 stars, based on 22625 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    91
    Thermo Fisher cdk6 complementary dna cdna
    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary <t>DNA</t> was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Cdk6 Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdk6 complementary dna cdna/product/Thermo Fisher
    Average 91 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    cdk6 complementary dna cdna - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    93
    Thermo Fisher transcriptomic complementary dna cdna
    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary <t>DNA</t> was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Transcriptomic Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transcriptomic complementary dna cdna/product/Thermo Fisher
    Average 93 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    transcriptomic complementary dna cdna - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    93
    Thermo Fisher complementary dna cdna template
    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary <t>DNA</t> was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p
    Complementary Dna Cdna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna template/product/Thermo Fisher
    Average 93 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna template - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    96
    Thermo Fisher complementary dna cdna synthesis
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 10175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna synthesis/product/Thermo Fisher
    Average 96 stars, based on 10175 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna synthesis - by Bioz Stars, 2020-05
    96/100 stars
      Buy from Supplier

    93
    Thermo Fisher complementary dna cdna conversion kit
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Complementary Dna Cdna Conversion Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna conversion kit/product/Thermo Fisher
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna conversion kit - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher verso complementary dna cdna kit
    <t>qRT-PCR</t> confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of <t>cDNA</t> generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.
    Verso Complementary Dna Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/verso complementary dna cdna kit/product/Thermo Fisher
    Average 99 stars, based on 905 article reviews
    Price from $9.99 to $1999.99
    verso complementary dna cdna kit - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    92
    Thermo Fisher complementary dna cdna synthesis supermix kit
    <t>qRT-PCR</t> confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of <t>cDNA</t> generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.
    Complementary Dna Cdna Synthesis Supermix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna synthesis supermix kit/product/Thermo Fisher
    Average 92 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna synthesis supermix kit - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    99
    Thermo Fisher double stranded complementary dna cdna
    <t>qRT-PCR</t> confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of <t>cDNA</t> generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.
    Double Stranded Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/double stranded complementary dna cdna/product/Thermo Fisher
    Average 99 stars, based on 251 article reviews
    Price from $9.99 to $1999.99
    double stranded complementary dna cdna - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher first strand complementary dna cdna
    <t>qRT-PCR</t> confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of <t>cDNA</t> generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.
    First Strand Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1964 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand complementary dna cdna/product/Thermo Fisher
    Average 96 stars, based on 1964 article reviews
    Price from $9.99 to $1999.99
    first strand complementary dna cdna - by Bioz Stars, 2020-05
    96/100 stars
      Buy from Supplier

    91
    Thermo Fisher complementary dna cdna master mix
    <t>qRT-PCR</t> confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of <t>cDNA</t> generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.
    Complementary Dna Cdna Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna cdna master mix/product/Thermo Fisher
    Average 91 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    complementary dna cdna master mix - by Bioz Stars, 2020-05
    91/100 stars
      Buy from Supplier

    89
    Thermo Fisher complementary dna oligonucleotides
    Activation of <t>Cdk5</t> by calpain contributes to <t>DNA</t> damage-induced ATM activation ( a ) Activation of ATM by Cdk5 mediated phosphorylation. HEK293 cells were transfected with Cdk5/p25 and ATM (wt, S794A and S794D mutants) as indicated. After 24 hours, ATM kinase activity was measured. The bottom panel shows the equal expression of ATM used in the lysates. The numbers are relative values with control set to one (same hereafter). ( b ) Activation of endogenous ATM by Cdk5/p25 or Cdk5/p35. Cdk5, p25 and p35 were overexpressed in HEK293 cells as indicated. After 24 hours, the levels of overexpressed proteins (top two panels) and ATM kinase activities (bottom panel) were determined. ( c ) Activation of Cdk5 and ATM by DNA damage. CGNs were treated with 10 μM camptothecin (CPT), 10 μM etoposide (Etop), 100 μg ml-1 bleomycin (Bleo), or 2 μM stausporine (STS) for 1 hour. Cdk5 and ATM kinase activity were measured using Histone H1 and PHAS-I as substrate, respectively. ( d ) CPT-induced p35 degradation. CGNs were treated with 10 μM CPT for the indicated periods of time. Level of cleaved α-fodrin, p35, p25 and Cdk5 were measured by immunoblotting. ( e ) Effects of inhibiting calpain on DNA damage-induced Cdk5 and ATM activation. CGNs were treated with or without 50 μM calpain inhibitor AK295 for 30 min and then with 10 μM CPT for the indicated periods of time. Cytoplamic and nuclear lysates were used for the determination of the levels of p35 and p25, and Cdk5 and ATM kinase activities. The membranes were reprobed for c-Raf, NeuN and actin as cytoplasmic fraction, nuclear fraction and loading controls, respectively.
    Complementary Dna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna oligonucleotides/product/Thermo Fisher
    Average 89 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    complementary dna oligonucleotides - by Bioz Stars, 2020-05
    89/100 stars
      Buy from Supplier

    85
    Thermo Fisher human mcm7 complementary dna cdna
    Pb/MCM7i13 transgenic construct. (A) Pb, prostate-specific rat probasin promoter ARR 2 PB; black box, HA tag; white box, Xpress tag; gray boxes, human <t>MCM7</t> coding sequence; red and blue boxes, miR-93/106b and miR-25 genes; yellow box, SV40 poly(A) (SV40 pA); orange box, probe used for Southern analysis of transgenic lines; green arrows, primers used for PCR analysis of transgenic lines and for genotyping. (B) Top: Southern blot analysis. Genomic <t>DNA</t> extracted from tails was digested with Bam HI and hybridized with a probe complementary to the SV40 poly(A) region as shown in (A). The Bam HI–digested Pb/MCM7i13 plasmid was used as positive control (C+). Bottom: PCR analysis. A forward primer complementary to the probasin promoter and a reverse primer complementary to the tag were used, as shown in (A). Empty Pb and Pb/MCM7i13 plasmids were used as negative (C−) and positive (C+) controls. L, 100-bp ladder. Three representative positive (P) and negative (N) mice are shown. (C) Abundance of the transgenic construct measured by real-time PCR in nine different lines. A primer pair specific for human MCM7 coding sequence was used. LE, ME, HE: low, medium, and high expressors, respectively. Human MCM7 .
    Human Mcm7 Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human mcm7 complementary dna cdna/product/Thermo Fisher
    Average 85 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    human mcm7 complementary dna cdna - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Thermo Fisher brain complementary dna
    Pb/MCM7i13 transgenic construct. (A) Pb, prostate-specific rat probasin promoter ARR 2 PB; black box, HA tag; white box, Xpress tag; gray boxes, human <t>MCM7</t> coding sequence; red and blue boxes, miR-93/106b and miR-25 genes; yellow box, SV40 poly(A) (SV40 pA); orange box, probe used for Southern analysis of transgenic lines; green arrows, primers used for PCR analysis of transgenic lines and for genotyping. (B) Top: Southern blot analysis. Genomic <t>DNA</t> extracted from tails was digested with Bam HI and hybridized with a probe complementary to the SV40 poly(A) region as shown in (A). The Bam HI–digested Pb/MCM7i13 plasmid was used as positive control (C+). Bottom: PCR analysis. A forward primer complementary to the probasin promoter and a reverse primer complementary to the tag were used, as shown in (A). Empty Pb and Pb/MCM7i13 plasmids were used as negative (C−) and positive (C+) controls. L, 100-bp ladder. Three representative positive (P) and negative (N) mice are shown. (C) Abundance of the transgenic construct measured by real-time PCR in nine different lines. A primer pair specific for human MCM7 coding sequence was used. LE, ME, HE: low, medium, and high expressors, respectively. Human MCM7 .
    Brain Complementary Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brain complementary dna/product/Thermo Fisher
    Average 85 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    brain complementary dna - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Thermo Fisher capacity complementary dna cdna archive kit
    The complementary <t>DNA</t> <t>(cDNA)</t> sequence of the junction of exon 12–13 in normal individuals and junction 12–14 in a Wilson Disease patient, where exon 13 has been entirely skipped.
    Capacity Complementary Dna Cdna Archive Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/capacity complementary dna cdna archive kit/product/Thermo Fisher
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    capacity complementary dna cdna archive kit - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    86
    Thermo Fisher abi high capacity complementary dna cdna kit
    The complementary <t>DNA</t> <t>(cDNA)</t> sequence of the junction of exon 12–13 in normal individuals and junction 12–14 in a Wilson Disease patient, where exon 13 has been entirely skipped.
    Abi High Capacity Complementary Dna Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/abi high capacity complementary dna cdna kit/product/Thermo Fisher
    Average 86 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    abi high capacity complementary dna cdna kit - by Bioz Stars, 2020-05
    86/100 stars
      Buy from Supplier

    93
    Thermo Fisher first strand complementary dna cdna synthesis
    The complementary <t>DNA</t> <t>(cDNA)</t> sequence of the junction of exon 12–13 in normal individuals and junction 12–14 in a Wilson Disease patient, where exon 13 has been entirely skipped.
    First Strand Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand complementary dna cdna synthesis/product/Thermo Fisher
    Average 93 stars, based on 505 article reviews
    Price from $9.99 to $1999.99
    first strand complementary dna cdna synthesis - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    Among VLP constituents, Vpx is necessary and sufficient to rescue HIV-1 from type I IFN . ( A ) MDDCs were treated with LPS for 24 hrs, then treated for 3 hrs with media or the indicated VSV-G-pseudotyped HIV-2 ROD or SIV MAC-251 VLPs, and finally challenged with a VSV-G-pseudotyped HIV-1 NL4-3 GFP reporter virus. Infectivity was measured by flow cytometry. ( B ) As indicated, 293T cells were co-transfected with a codon optimized SIV MAC251 vpx expression plasmid and HIV-1 GFP reporter vectors bearing either wild-type Gag or Gag with an engineered Vpx binding motif (DPAVDLL). Proteins from the cell lysate and from virion preparations were separated by SDS-PAGE and then immunoblotted with anti-Vpx or anti-p24 antibodies. ( C ) MDDCs treated with IFN-β for 24 h and were then challenged with VSV-G-pseudotyped HIV-1 GFP reporter vectors with wild-type HIV-1 Gag or HIV-1 Gag bearing the engineered Vpx binding motif (DPAVDLL). Both HIV-1 reporter vectors were produced in the presence of empty pcDNA3.1 plasmid or pcDNA3.1 containing a codon-optimized SIV MAC-251 vpx cDNA. Data are representative of one of at least three independent experiments. Error bars represent ± SD (n = 3).

    Journal: Retrovirology

    Article Title: Vpx rescues HIV-1 transduction of dendritic cells from the antiviral state established by type 1 interferon

    doi: 10.1186/1742-4690-8-49

    Figure Lengend Snippet: Among VLP constituents, Vpx is necessary and sufficient to rescue HIV-1 from type I IFN . ( A ) MDDCs were treated with LPS for 24 hrs, then treated for 3 hrs with media or the indicated VSV-G-pseudotyped HIV-2 ROD or SIV MAC-251 VLPs, and finally challenged with a VSV-G-pseudotyped HIV-1 NL4-3 GFP reporter virus. Infectivity was measured by flow cytometry. ( B ) As indicated, 293T cells were co-transfected with a codon optimized SIV MAC251 vpx expression plasmid and HIV-1 GFP reporter vectors bearing either wild-type Gag or Gag with an engineered Vpx binding motif (DPAVDLL). Proteins from the cell lysate and from virion preparations were separated by SDS-PAGE and then immunoblotted with anti-Vpx or anti-p24 antibodies. ( C ) MDDCs treated with IFN-β for 24 h and were then challenged with VSV-G-pseudotyped HIV-1 GFP reporter vectors with wild-type HIV-1 Gag or HIV-1 Gag bearing the engineered Vpx binding motif (DPAVDLL). Both HIV-1 reporter vectors were produced in the presence of empty pcDNA3.1 plasmid or pcDNA3.1 containing a codon-optimized SIV MAC-251 vpx cDNA. Data are representative of one of at least three independent experiments. Error bars represent ± SD (n = 3).

    Article Snippet: First-strand cDNA was generated using the SuperScript III First-Strand Synthesis System (Invitrogen) using 2 μg total RNA and random hexamers, according to the manufacturer's protocol.

    Techniques: Infection, Flow Cytometry, Cytometry, Transfection, Expressing, Plasmid Preparation, Binding Assay, SDS Page, Produced

    miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

    Journal: Cancer Cell International

    Article Title: MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells

    doi: 10.1186/s12935-017-0500-0

    Figure Lengend Snippet: miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

    Article Snippet: One microgram of total RNA was used to generate complementary DNA (cDNA) by superscript reverse transcriptase (Invitrogen).

    Techniques: Generated, Quantitative RT-PCR, Expressing, Transfection, Incubation, MTT Assay

    Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Chromatin Immunoprecipitation, Microarray, Sequencing, Expressing, Transformation Assay, Produced

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced, Amplification, Immunohistochemistry

    Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Sequencing, Expressing, Microarray

    Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray

    Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced

    Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Journal: BMC Bioinformatics

    Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

    doi: 10.1186/1471-2105-6-107

    Figure Lengend Snippet: Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

    Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

    Techniques: Sequencing, Microarray

    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Identification of a host collagen inducing factor from the excretory secretory proteins of Trichinella spiralis

    doi: 10.1371/journal.pntd.0006516

    Figure Lengend Snippet: Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Article Snippet: Real-time PCR Homogenized ear tissues were mixed with TRIzol (Invitrogen, Germany), and total RNA extraction and cDNA synthesis (Invitrogen, Germany) was performed in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Mouse Assay, Construct, Real-time Polymerase Chain Reaction

    Southern and Northern analysis of expressed X-linked sequences and cDNA clones. ( A ) A 2.2-kb genomic fragment, FIJG, was hybridized to a Southern blot containing DNA digested with Hin dIII and either Xba I or Bgl ). This probe detects two loci in genomic DNA, one of which maps distal to the t(X;17) breakpoint (5.5- and 3.3-kb bands, referred to as FIJG-4), whereas the other maps proximal to the breakpoint (8.0-kb band, referred to as FIJG-5). The probe is colinear with genomic DNA and does not contain Hin dIII, Xba I, or Bgl II restriction sites. ( B ) Expression patterns were examined on a multiple-tissue Northern blot (CLONTECH) by using the same 2.2-kb genomic probe. A transcript of ≈1.3 kb was detected in skeletal muscle tissue only. ( C ) Secondary selected cDNA products from a typical direct cDNA selection experiment were used as a complex probe and hybridized to Eco RI digests of a partial cosmid contig covering the region that was selected (lanes 1–17). Hybridizing fragments correspond to genomic fragments homologous to selected cDNA clones. ( D ) The selected cDNA clone ADS9 is widely expressed and detects on Northern blots a single RNA species of ≈1.0 kb in various tissues.

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

    Article Title: Chromosomal basis of X chromosome inactivation: Identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation

    doi:

    Figure Lengend Snippet: Southern and Northern analysis of expressed X-linked sequences and cDNA clones. ( A ) A 2.2-kb genomic fragment, FIJG, was hybridized to a Southern blot containing DNA digested with Hin dIII and either Xba I or Bgl ). This probe detects two loci in genomic DNA, one of which maps distal to the t(X;17) breakpoint (5.5- and 3.3-kb bands, referred to as FIJG-4), whereas the other maps proximal to the breakpoint (8.0-kb band, referred to as FIJG-5). The probe is colinear with genomic DNA and does not contain Hin dIII, Xba I, or Bgl II restriction sites. ( B ) Expression patterns were examined on a multiple-tissue Northern blot (CLONTECH) by using the same 2.2-kb genomic probe. A transcript of ≈1.3 kb was detected in skeletal muscle tissue only. ( C ) Secondary selected cDNA products from a typical direct cDNA selection experiment were used as a complex probe and hybridized to Eco RI digests of a partial cosmid contig covering the region that was selected (lanes 1–17). Hybridizing fragments correspond to genomic fragments homologous to selected cDNA clones. ( D ) The selected cDNA clone ADS9 is widely expressed and detects on Northern blots a single RNA species of ≈1.0 kb in various tissues.

    Article Snippet: Approximately 5 μg of poly(A)+ RNA was converted to double-stranded cDNA by using a cDNA synthesis system (GIBCO/BRL).

    Techniques: Northern Blot, Clone Assay, Southern Blot, Expressing, Selection

    qRT-PCR confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of cDNA generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.

    Journal: Molecular and Cellular Neurosciences

    Article Title: In vitro and intrathecal siRNA mediated KV1.1 knock-down in primary sensory neurons

    doi: 10.1016/j.mcn.2011.08.007

    Figure Lengend Snippet: qRT-PCR confirms knock-down of K V 1.1 following transfection with 3 sequence specific mRNAs. The amount of cDNA generated from the K V 1.1 mRNA at 2 days following transfection was normalised to β-actin, mean fall in K V 1.1 mRNA = 29. 67 ± 2.11%; ** p = 0.005, one-sample t -test, indicative of a minimum 43% mRNA knock-down in neurons transfected with active sequence.

    Article Snippet: RNA (1 μg) was reverse transcribed with a cDNA synthesis kit (Invitrogen) and expression levels measured by qRT-PCR on a StepOnePlus machine (Applied Biosystems), utilising Fast SYBR Green Master Mix (Applied Biosystems).

    Techniques: Quantitative RT-PCR, Transfection, Sequencing, Generated

    Activation of Cdk5 by calpain contributes to DNA damage-induced ATM activation ( a ) Activation of ATM by Cdk5 mediated phosphorylation. HEK293 cells were transfected with Cdk5/p25 and ATM (wt, S794A and S794D mutants) as indicated. After 24 hours, ATM kinase activity was measured. The bottom panel shows the equal expression of ATM used in the lysates. The numbers are relative values with control set to one (same hereafter). ( b ) Activation of endogenous ATM by Cdk5/p25 or Cdk5/p35. Cdk5, p25 and p35 were overexpressed in HEK293 cells as indicated. After 24 hours, the levels of overexpressed proteins (top two panels) and ATM kinase activities (bottom panel) were determined. ( c ) Activation of Cdk5 and ATM by DNA damage. CGNs were treated with 10 μM camptothecin (CPT), 10 μM etoposide (Etop), 100 μg ml-1 bleomycin (Bleo), or 2 μM stausporine (STS) for 1 hour. Cdk5 and ATM kinase activity were measured using Histone H1 and PHAS-I as substrate, respectively. ( d ) CPT-induced p35 degradation. CGNs were treated with 10 μM CPT for the indicated periods of time. Level of cleaved α-fodrin, p35, p25 and Cdk5 were measured by immunoblotting. ( e ) Effects of inhibiting calpain on DNA damage-induced Cdk5 and ATM activation. CGNs were treated with or without 50 μM calpain inhibitor AK295 for 30 min and then with 10 μM CPT for the indicated periods of time. Cytoplamic and nuclear lysates were used for the determination of the levels of p35 and p25, and Cdk5 and ATM kinase activities. The membranes were reprobed for c-Raf, NeuN and actin as cytoplasmic fraction, nuclear fraction and loading controls, respectively.

    Journal: Nature cell biology

    Article Title: Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death

    doi: 10.1038/ncb1829

    Figure Lengend Snippet: Activation of Cdk5 by calpain contributes to DNA damage-induced ATM activation ( a ) Activation of ATM by Cdk5 mediated phosphorylation. HEK293 cells were transfected with Cdk5/p25 and ATM (wt, S794A and S794D mutants) as indicated. After 24 hours, ATM kinase activity was measured. The bottom panel shows the equal expression of ATM used in the lysates. The numbers are relative values with control set to one (same hereafter). ( b ) Activation of endogenous ATM by Cdk5/p25 or Cdk5/p35. Cdk5, p25 and p35 were overexpressed in HEK293 cells as indicated. After 24 hours, the levels of overexpressed proteins (top two panels) and ATM kinase activities (bottom panel) were determined. ( c ) Activation of Cdk5 and ATM by DNA damage. CGNs were treated with 10 μM camptothecin (CPT), 10 μM etoposide (Etop), 100 μg ml-1 bleomycin (Bleo), or 2 μM stausporine (STS) for 1 hour. Cdk5 and ATM kinase activity were measured using Histone H1 and PHAS-I as substrate, respectively. ( d ) CPT-induced p35 degradation. CGNs were treated with 10 μM CPT for the indicated periods of time. Level of cleaved α-fodrin, p35, p25 and Cdk5 were measured by immunoblotting. ( e ) Effects of inhibiting calpain on DNA damage-induced Cdk5 and ATM activation. CGNs were treated with or without 50 μM calpain inhibitor AK295 for 30 min and then with 10 μM CPT for the indicated periods of time. Cytoplamic and nuclear lysates were used for the determination of the levels of p35 and p25, and Cdk5 and ATM kinase activities. The membranes were reprobed for c-Raf, NeuN and actin as cytoplasmic fraction, nuclear fraction and loading controls, respectively.

    Article Snippet: Cdk5 shRNA lentivirus and infection To construct the 19 nucleotide (corresponding to rat Cdk5 nt positions 639-657) hairpin siRNA cassettes, two complementary DNA oligonucleotides were chemically synthesized in Invitrogen, annealed, and inserted into pSUPER shuttle vector immediately downstream of the H1 RNA pol III promoter: 5’-GATCCCC- GAGGATCTTTCGACTGCTA-TTCAAGAGA-TAGCAGTCGAAAGATCCTC-TTTTTGGAAA-3 ’ a n d 5 ’-AGCTTTTCCAAAAA-GAGGATCTTTCGACTGCTA-TCTCTTGAA- TAGCAGTCGAAAGATCCTC-GGG-3’.

    Techniques: Activation Assay, Transfection, Activity Assay, Expressing, Cycling Probe Technology

    Cdk5-ATM signal regulates DNA damage-induced neuronal death ( a ) Inhibition of CPT-induced neuronal death by roscovitine. CGNs were exposed to 10 μM CPT and different concentrations of roscovitine for 24 hours. Neuronal viability was measured by WST-1 assay. ( b ) Time dependent effect of roscovitine on CPT-induced neuronal death. CGNs were treated 10 μM CPT alone or with 10μM roscovitine (Ros) or 10 μM Ku-55933 (KU) for the different periods of time. WST-1 data are mean ± SD (n =4. **, p

    Journal: Nature cell biology

    Article Title: Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death

    doi: 10.1038/ncb1829

    Figure Lengend Snippet: Cdk5-ATM signal regulates DNA damage-induced neuronal death ( a ) Inhibition of CPT-induced neuronal death by roscovitine. CGNs were exposed to 10 μM CPT and different concentrations of roscovitine for 24 hours. Neuronal viability was measured by WST-1 assay. ( b ) Time dependent effect of roscovitine on CPT-induced neuronal death. CGNs were treated 10 μM CPT alone or with 10μM roscovitine (Ros) or 10 μM Ku-55933 (KU) for the different periods of time. WST-1 data are mean ± SD (n =4. **, p

    Article Snippet: Cdk5 shRNA lentivirus and infection To construct the 19 nucleotide (corresponding to rat Cdk5 nt positions 639-657) hairpin siRNA cassettes, two complementary DNA oligonucleotides were chemically synthesized in Invitrogen, annealed, and inserted into pSUPER shuttle vector immediately downstream of the H1 RNA pol III promoter: 5’-GATCCCC- GAGGATCTTTCGACTGCTA-TTCAAGAGA-TAGCAGTCGAAAGATCCTC-TTTTTGGAAA-3 ’ a n d 5 ’-AGCTTTTCCAAAAA-GAGGATCTTTCGACTGCTA-TCTCTTGAA- TAGCAGTCGAAAGATCCTC-GGG-3’.

    Techniques: Inhibition, Cycling Probe Technology, WST-1 Assay

    Inhibition of Cdk5 blocks DNA damage-induced phosphorylation and activation of ATM and its effects on downstream targets ( a ) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by roscovitine. Neurons differentiated from SH-SY5Y cells were pretreated with or without 10 μM roscovitine (Ros) for 30 min and followed with 10 μM CPT for the indicated periods of time. Phospho-S794, phospho-S1981 and total ATM were determined by immunoblotting. ( b ) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by knocking down Cdk5. Neurons differentiated from SH-SY5Y cells were infected with control or Cdk5 RNAi adenovirus for 24 hours, and then treated with 10 μM CPT for the indicated periods of time. Cdk5, phospho-S794, phospho-S1981, total ATM and actin were determined by immunoblotting. ( c ) Inhibition of CPT-induced ATM activation and p53 phosphorylation by roscovitine. CGNs were treated with or without 10 μM Roscovitine for 30 min and then with 10 μM CPT treatment for 1 hour. ATM kinase activity, phospho-S15, total p53 and actin were measured in the same set of lysates. ( d ) Inhibition of CPT-induced ATM activation and p53 phosphorylation by knocking down Cdk5. CGNs were infected with Cdk5i or scrambled RNAi lentivirus for 72 hours and then treated with 10 μM CPT treatment for 1 hour. Cdk5 and ATM kinase activities and levels for Cdk5, phospho-S15, total p53 and actin were measured in the same set of lysates. ( e ) Reduction of CPT-induced γ-H2AX foci formation by roscovitine. CGNs were treated with or without 10 μM Roscovitine (Ros) for 30 min and then exposed to 10 μM CPT for 1 hour. γ-H2AX (green) was detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The average numbers of foci/cell counted blindly are control, 0.26; CPT, 3.91; CPT+Ros, 1.08 (p

    Journal: Nature cell biology

    Article Title: Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death

    doi: 10.1038/ncb1829

    Figure Lengend Snippet: Inhibition of Cdk5 blocks DNA damage-induced phosphorylation and activation of ATM and its effects on downstream targets ( a ) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by roscovitine. Neurons differentiated from SH-SY5Y cells were pretreated with or without 10 μM roscovitine (Ros) for 30 min and followed with 10 μM CPT for the indicated periods of time. Phospho-S794, phospho-S1981 and total ATM were determined by immunoblotting. ( b ) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by knocking down Cdk5. Neurons differentiated from SH-SY5Y cells were infected with control or Cdk5 RNAi adenovirus for 24 hours, and then treated with 10 μM CPT for the indicated periods of time. Cdk5, phospho-S794, phospho-S1981, total ATM and actin were determined by immunoblotting. ( c ) Inhibition of CPT-induced ATM activation and p53 phosphorylation by roscovitine. CGNs were treated with or without 10 μM Roscovitine for 30 min and then with 10 μM CPT treatment for 1 hour. ATM kinase activity, phospho-S15, total p53 and actin were measured in the same set of lysates. ( d ) Inhibition of CPT-induced ATM activation and p53 phosphorylation by knocking down Cdk5. CGNs were infected with Cdk5i or scrambled RNAi lentivirus for 72 hours and then treated with 10 μM CPT treatment for 1 hour. Cdk5 and ATM kinase activities and levels for Cdk5, phospho-S15, total p53 and actin were measured in the same set of lysates. ( e ) Reduction of CPT-induced γ-H2AX foci formation by roscovitine. CGNs were treated with or without 10 μM Roscovitine (Ros) for 30 min and then exposed to 10 μM CPT for 1 hour. γ-H2AX (green) was detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The average numbers of foci/cell counted blindly are control, 0.26; CPT, 3.91; CPT+Ros, 1.08 (p

    Article Snippet: Cdk5 shRNA lentivirus and infection To construct the 19 nucleotide (corresponding to rat Cdk5 nt positions 639-657) hairpin siRNA cassettes, two complementary DNA oligonucleotides were chemically synthesized in Invitrogen, annealed, and inserted into pSUPER shuttle vector immediately downstream of the H1 RNA pol III promoter: 5’-GATCCCC- GAGGATCTTTCGACTGCTA-TTCAAGAGA-TAGCAGTCGAAAGATCCTC-TTTTTGGAAA-3 ’ a n d 5 ’-AGCTTTTCCAAAAA-GAGGATCTTTCGACTGCTA-TCTCTTGAA- TAGCAGTCGAAAGATCCTC-GGG-3’.

    Techniques: Inhibition, Activation Assay, Cycling Probe Technology, Infection, Activity Assay, Immunocytochemistry, Labeling

    Pb/MCM7i13 transgenic construct. (A) Pb, prostate-specific rat probasin promoter ARR 2 PB; black box, HA tag; white box, Xpress tag; gray boxes, human MCM7 coding sequence; red and blue boxes, miR-93/106b and miR-25 genes; yellow box, SV40 poly(A) (SV40 pA); orange box, probe used for Southern analysis of transgenic lines; green arrows, primers used for PCR analysis of transgenic lines and for genotyping. (B) Top: Southern blot analysis. Genomic DNA extracted from tails was digested with Bam HI and hybridized with a probe complementary to the SV40 poly(A) region as shown in (A). The Bam HI–digested Pb/MCM7i13 plasmid was used as positive control (C+). Bottom: PCR analysis. A forward primer complementary to the probasin promoter and a reverse primer complementary to the tag were used, as shown in (A). Empty Pb and Pb/MCM7i13 plasmids were used as negative (C−) and positive (C+) controls. L, 100-bp ladder. Three representative positive (P) and negative (N) mice are shown. (C) Abundance of the transgenic construct measured by real-time PCR in nine different lines. A primer pair specific for human MCM7 coding sequence was used. LE, ME, HE: low, medium, and high expressors, respectively. Human MCM7 .

    Journal: Science signaling

    Article Title: Identification of the miR-106b~25 MicroRNA Cluster as a Proto-Oncogenic PTEN-Targeting Intron That Cooperates with Its Host Gene MCM7 in Transformation

    doi: 10.1126/scisignal.2000594

    Figure Lengend Snippet: Pb/MCM7i13 transgenic construct. (A) Pb, prostate-specific rat probasin promoter ARR 2 PB; black box, HA tag; white box, Xpress tag; gray boxes, human MCM7 coding sequence; red and blue boxes, miR-93/106b and miR-25 genes; yellow box, SV40 poly(A) (SV40 pA); orange box, probe used for Southern analysis of transgenic lines; green arrows, primers used for PCR analysis of transgenic lines and for genotyping. (B) Top: Southern blot analysis. Genomic DNA extracted from tails was digested with Bam HI and hybridized with a probe complementary to the SV40 poly(A) region as shown in (A). The Bam HI–digested Pb/MCM7i13 plasmid was used as positive control (C+). Bottom: PCR analysis. A forward primer complementary to the probasin promoter and a reverse primer complementary to the tag were used, as shown in (A). Empty Pb and Pb/MCM7i13 plasmids were used as negative (C−) and positive (C+) controls. L, 100-bp ladder. Three representative positive (P) and negative (N) mice are shown. (C) Abundance of the transgenic construct measured by real-time PCR in nine different lines. A primer pair specific for human MCM7 coding sequence was used. LE, ME, HE: low, medium, and high expressors, respectively. Human MCM7 .

    Article Snippet: The following antibodies and reagents were used: antibodies against Hsp-90 #61041 (Becton Dickinson); PTEN #9559 (WB), pAkt (Ser473 ) #9271 (WB), #3787 (IHC), Akt #9272, pS6 #2211 (Cell Signaling); DICER #CG006 (Clonegene); MSCV-neo (Clontech); siGENOME non-targeting small interfering RNA (siRNA) #2 (si-Luc), si-miR-19a, si-miR-19b, si-miR-22, si-miR-25, si-miR-92a, si-miR-17, si-miR-20a, si-miR-93, si-miR-106b, si-miR-302a, si-miR-372, si-miR-373, si-PTEN, siGLO RISC-free control siRNA miRNA antisense inhibitor negative control #1, miR-19a antisense inhibitor, miR-22 antisense inhibitor, miR-25 antisense inhibitor, miR-93 antisense inhibitor, miR-106b antisense inhibitor, Dharmafect 1 (Dharmacon); miR-17, miR-19a, miR-20a, miR-22, miR-25, miR-93, and miR-106b 3′ digoxigenin (DIG)–labeled LNA ISH probes (Exiqon); lipofectamine 2000, Trizol reagent, deoxyribonuclease I (DNase I) amplification grade, SuperScript II reverse transcriptase, Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, fetal bovine serum (FBS), keratinocyte serum-free medium, epidermal growth factor (EGF), bovine pituitary extract (BPE), antibody against Xpress Tag, pcDNA3.1/His C, geneticin (G418), hygromycin, antibody against PTEN (IHC) #51-2400 (Invitrogen); human MCM7 complementary DNA (cDNA) #MHS1011-75176 (Open Biosystems); pGL3-Control ( Firefly luciferase ), pRL-TK ( Renilla luciferase ), Dual-Luciferase reporter assay (Promega); fraction V bovine serum albumin (BSA), polybrene, insulin, antibody against actin #A3853, anti-FLAG antibody #F3165, puromycin (Sigma); QuantiTect Sybr Green PCRkit (Qiagen); antibody against MCM7 #9966 (Santa Cruz Biotechnology); QuikChange II XL Site-Directed Mutagenesis Kit, Herculase Taq polymerase (Stratagene); Ki67 antibody #VP-K451 (Vector Laboratories).

    Techniques: Transgenic Assay, Construct, Sequencing, Polymerase Chain Reaction, Southern Blot, Plasmid Preparation, Positive Control, Mouse Assay, Real-time Polymerase Chain Reaction

    The complementary DNA (cDNA) sequence of the junction of exon 12–13 in normal individuals and junction 12–14 in a Wilson Disease patient, where exon 13 has been entirely skipped.

    Journal: Sultan Qaboos University Medical Journal

    Article Title: A Novel Splice-site Allelic Variant is Responsible for Wilson Disease in an Omani Family

    doi:

    Figure Lengend Snippet: The complementary DNA (cDNA) sequence of the junction of exon 12–13 in normal individuals and junction 12–14 in a Wilson Disease patient, where exon 13 has been entirely skipped.

    Article Snippet: Reverse transcription of DNA was performed using the high capacity complementary DNA (cDNA) Archive kit (Applied Biosystems, USA).

    Techniques: Sequencing