nested-pcr strategy Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher gateway pcr cloning strategy
    Role of murine SERPINB1 isoforms in <t>caspase-1/–11</t> inhibition. a , The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P 2 –P 1 –P 1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b , Verification of Serpinb1 -targeting shRNA’s silencing efficiency by <t>qRT-PCR</t> in DC2.4 cells. mRNA expression was normalized to 18S , and fold change was calculated relative to scramble. c , Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a , Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d , Caspase-1 activation in Serpinb1a −/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e , f , IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1 −/− Casp11 −/− BMDMs were transduced with scrambled or pan- Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b , f and from n = 7 per group, pooled from two independent experiments in d , and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e . P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b , by an two-tailed unpaired t -test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e , f .
    Gateway Pcr Cloning Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gateway pcr cloning strategy/product/Thermo Fisher
    Average 99 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    gateway pcr cloning strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    Millipore rt pcr strategy
    Role of murine SERPINB1 isoforms in <t>caspase-1/–11</t> inhibition. a , The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P 2 –P 1 –P 1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b , Verification of Serpinb1 -targeting shRNA’s silencing efficiency by <t>qRT-PCR</t> in DC2.4 cells. mRNA expression was normalized to 18S , and fold change was calculated relative to scramble. c , Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a , Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d , Caspase-1 activation in Serpinb1a −/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e , f , IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1 −/− Casp11 −/− BMDMs were transduced with scrambled or pan- Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b , f and from n = 7 per group, pooled from two independent experiments in d , and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e . P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b , by an two-tailed unpaired t -test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e , f .
    Rt Pcr Strategy, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rt pcr strategy/product/Millipore
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    rt pcr strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    89
    Stratagene pcr strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr strategy/product/Stratagene
    Average 89 stars, based on 133 article reviews
    Price from $9.99 to $1999.99
    pcr strategy - by Bioz Stars, 2020-12
    89/100 stars
      Buy from Supplier

    88
    Stratagene quikchange pcr strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Quikchange Pcr Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange pcr strategy/product/Stratagene
    Average 88 stars, based on 39 article reviews
    Price from $9.99 to $1999.99
    quikchange pcr strategy - by Bioz Stars, 2020-12
    88/100 stars
      Buy from Supplier

    86
    Stratagene polymerase chain reaction based mutagenesis strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Polymerase Chain Reaction Based Mutagenesis Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polymerase chain reaction based mutagenesis strategy/product/Stratagene
    Average 86 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    polymerase chain reaction based mutagenesis strategy - by Bioz Stars, 2020-12
    86/100 stars
      Buy from Supplier

    86
    Stratagene pcr based quikchangetm strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based Quikchangetm Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based quikchangetm strategy/product/Stratagene
    Average 86 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    pcr based quikchangetm strategy - by Bioz Stars, 2020-12
    86/100 stars
      Buy from Supplier

    85
    Seegene multiplex pcr strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Multiplex Pcr Strategy, supplied by Seegene, used in various techniques. Bioz Stars score: 85/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/multiplex pcr strategy/product/Seegene
    Average 85 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    multiplex pcr strategy - by Bioz Stars, 2020-12
    85/100 stars
      Buy from Supplier

    99
    Thermo Fisher real time pcr rt qpcr strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Real Time Pcr Rt Qpcr Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time pcr rt qpcr strategy/product/Thermo Fisher
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    real time pcr rt qpcr strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher attb adapter pcr strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Attb Adapter Pcr Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/attb adapter pcr strategy/product/Thermo Fisher
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    attb adapter pcr strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    97
    Thermo Fisher genome wide pcr amplification strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Genome Wide Pcr Amplification Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genome wide pcr amplification strategy/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    genome wide pcr amplification strategy - by Bioz Stars, 2020-12
    97/100 stars
      Buy from Supplier

    93
    Cellecta two step pcr amplification strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Two Step Pcr Amplification Strategy, supplied by Cellecta, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/two step pcr amplification strategy/product/Cellecta
    Average 93 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    two step pcr amplification strategy - by Bioz Stars, 2020-12
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcr based 2 step strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based 2 Step Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based 2 step strategy/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pcr based 2 step strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    93
    Millipore pcr based cloning strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based Cloning Strategy, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based cloning strategy/product/Millipore
    Average 93 stars, based on 96 article reviews
    Price from $9.99 to $1999.99
    pcr based cloning strategy - by Bioz Stars, 2020-12
    93/100 stars
      Buy from Supplier

    88
    Stratagene pcr based cloning strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based Cloning Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based cloning strategy/product/Stratagene
    Average 88 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pcr based cloning strategy - by Bioz Stars, 2020-12
    88/100 stars
      Buy from Supplier

    93
    Addgene inc pcr based strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based Strategy, supplied by Addgene inc, 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/pcr based strategy/product/Addgene inc
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pcr based strategy - by Bioz Stars, 2020-12
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcr based strategy
    <t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
    Pcr Based Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based strategy/product/Thermo Fisher
    Average 99 stars, based on 119 article reviews
    Price from $9.99 to $1999.99
    pcr based strategy - by Bioz Stars, 2020-12
    99/100 stars
      Buy from Supplier

    Image Search Results


    Role of murine SERPINB1 isoforms in caspase-1/–11 inhibition. a , The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P 2 –P 1 –P 1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b , Verification of Serpinb1 -targeting shRNA’s silencing efficiency by qRT-PCR in DC2.4 cells. mRNA expression was normalized to 18S , and fold change was calculated relative to scramble. c , Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a , Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d , Caspase-1 activation in Serpinb1a −/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e , f , IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1 −/− Casp11 −/− BMDMs were transduced with scrambled or pan- Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b , f and from n = 7 per group, pooled from two independent experiments in d , and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e . P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b , by an two-tailed unpaired t -test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e , f .

    Journal: Nature immunology

    Article Title: SERPINB1-mediated checkpoint of inflammatory caspase activation

    doi: 10.1038/s41590-018-0303-z

    Figure Lengend Snippet: Role of murine SERPINB1 isoforms in caspase-1/–11 inhibition. a , The RCL and CBM sequence alignment of human SERPINB1 and mouse SERPINB1a, SERPINB1b and SERPINB1c. The conserved P 2 –P 1 –P 1 residues in the RCL sequence are indicated by orange font, and the CBM sequence is shown in red font. The conserved 13 aa in the CBM sequence are indicated in the red-dotted box. Specified amino acid numbers are based on human SERPINB1. b , Verification of Serpinb1 -targeting shRNA’s silencing efficiency by qRT-PCR in DC2.4 cells. mRNA expression was normalized to 18S , and fold change was calculated relative to scramble. c , Immunoblots of cleaved p10 subunit of caspase-1 in DC2.4 cells after Serpinb1a , Serpinb1b and/or Serpinb1c depletion. WCLs and SUPs were obtained at 48 h post-transduction and were immunoblotted with indicated antibodies. d , Caspase-1 activation in Serpinb1a −/− pPMNs. FLICA-positive staining was analyzed by flow cytometry. e , f , IL-1β secretion and cell death after Serpinb1 depletion in BMDMs. Wild-type (WT) and Casp1 −/− Casp11 −/− BMDMs were transduced with scrambled or pan- Serpinb1 shRNA lentivirus. Cytokines were quantified by enzyme-linked immunosorbent assay (ELISA), and cell viability was determined by ATP-based assay. Data in c are representative of two independent experiments. Data are presented as mean ± s.e.m. from n = 3 independent experiments in b , f and from n = 7 per group, pooled from two independent experiments in d , and as box and whiskers (min to max) from n = 6 pooled from three independent experiments in e . P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in b , by an two-tailed unpaired t -test in d and by two-way ANOVA with Bonferroni’s comparison relative to scramble in e , f .

    Article Snippet: SERPINB1 truncation, deletion and point mutants, the caspase-4 CARD point mutant and caspase-11-C254 A were constructed by a standard PCR cloning strategy or a GeneArt Site-Directed mutagenesis system (Invitrogen).

    Techniques: Inhibition, Sequencing, shRNA, Quantitative RT-PCR, Expressing, Western Blot, Transduction, Activation Assay, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, ATP Assay, Two Tailed Test

    HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Journal: Retrovirology

    Article Title: High-throughput profiling of point mutations across the HIV-1 genome

    doi: 10.1186/s12977-014-0124-6

    Figure Lengend Snippet: HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Article Snippet: Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification.

    Techniques: Mutagenesis, Next-Generation Sequencing, Sample Prep, Polymerase Chain Reaction, Amplification, Concentration Assay

    Changes in PGC-1α expression and acetylation after chronic exercise. A : Real-time PCR analyses of PGC-1 α mRNA expression in the skeletal muscle of C57BL/6J wild-type mice (black columns) and ob / ob mice (gray columns). All data are normalized per 18S rRNA. B : Densitometry of protein data. Homogenates of skeletal muscle were probed with an antibody detecting PGC-1α (90 kDa). Blots were also probed with GAPDH as a loading control. C : Densitometry of immunoprecipitation (IP) experiments performed on skeletal muscle lysates, using PGC-1α for precipitation and an antibody directed against acetyl-lysine or PGC-1α for detection. Data are given as acetylated PGC-1α per total PGC-1α in these samples. All data are means ± SEM. * P

    Journal: Diabetes

    Article Title: Mitochondrial Biogenesis and Peroxisome Proliferator-Activated Receptor-? Coactivator-1? (PGC-1?) Deacetylation by Physical Activity

    doi: 10.2337/db10-0331

    Figure Lengend Snippet: Changes in PGC-1α expression and acetylation after chronic exercise. A : Real-time PCR analyses of PGC-1 α mRNA expression in the skeletal muscle of C57BL/6J wild-type mice (black columns) and ob / ob mice (gray columns). All data are normalized per 18S rRNA. B : Densitometry of protein data. Homogenates of skeletal muscle were probed with an antibody detecting PGC-1α (90 kDa). Blots were also probed with GAPDH as a loading control. C : Densitometry of immunoprecipitation (IP) experiments performed on skeletal muscle lysates, using PGC-1α for precipitation and an antibody directed against acetyl-lysine or PGC-1α for detection. Data are given as acetylated PGC-1α per total PGC-1α in these samples. All data are means ± SEM. * P

    Article Snippet: Site-directed mutations at Thr177 and Ser538 in PGC-1α were introduced by a PCR-based strategy with the QuikChange site-directed mutagenesis kit (Stratagene).

    Techniques: Pyrolysis Gas Chromatography, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Immunoprecipitation