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  • 99
    Integrated DNA Technologies target specific gblock gene fragments
    Target Specific Gblock Gene Fragments, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher 3730xl dna analyzer
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 48698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    3730xl dna analyzer - by Bioz Stars, 2020-07
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    99
    Millipore target dna fragment
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Target Dna Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna fragments target
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Dna Fragments Target, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc target dna fragments
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Target Dna Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Geneaid Biotech Ltd target dna fragments
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Target Dna Fragments, supplied by Geneaid Biotech Ltd, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia target dna fragment
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Target Dna Fragment, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Double Helix single strand dna fragments target
    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 <t>amplicons</t> could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .
    Single Strand Dna Fragments Target, supplied by Double Helix, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher target dna fragment wtih nfκb p65 transcription factor assay kit
    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
    Target Dna Fragment Wtih Nfκb P65 Transcription Factor Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher target cdna fragments
    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
    Target Cdna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co dna purification kit
    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
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    Thermo Fisher abi sequencer 3730 dna analyzer
    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
    Abi Sequencer 3730 Dna Analyzer, 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
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    Beyotime dna purification kit
    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
    Dna Purification Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 93/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
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    Autophagy enhances <t>NFκB</t> activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB <t>p65</t> (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in <t>DNA</t> was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p
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    Image Search Results


    Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 amplicons could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .

    Journal: PLoS ONE

    Article Title: Atlas of tissue- and developmental stage specific gene expression for the bovine insulin-like growth factor (IGF) system

    doi: 10.1371/journal.pone.0200466

    Figure Lengend Snippet: Bovine IGF2 gene and transcript structure with primer locations for amplification of promoter specific transcripts and splice variants. The exon-intron structure of bovine insulin/insulin-like growth factor 2 ( INS/IGF2 , GenBank accession no. EU518675.1) with locations of five promoters (P0, P1, P2, P3 and P4) is shown at the top with promoters ( IGF2 -P0 –P4) and splice variant specific transcripts indicated below. Red and green boxes depict untranslated and protein coding exons, respectively. Forward (F) and reverse (R) primers are indicated with region spanned, including intron where applicable, symbolized by a black bar between primers above the transcript. According to the transcription initiation site of human IGF2 -P0 transcript, the putative orthologous bovine transcript is predicted to originate from a highly conserved region located upstream of the splice donor site of transcript P1 exon 2. We could specifically amplify bovine IGF2 -P0 using a strategically designed forward primer within this unique 5’-UTR sequence and the reverse primer located within exon 2. The two splice variants of P1 promoter transcripts include leading exon 1 which is alternatively spliced onto exons 2 and 3 ( IGF2 -P1e2) and exon 3 ( IGF2 -P1e3) plus the coding exons. In order to amplify the P1 promoter transcripts, two pairs of primers located within exon 2 (for IGF2 -P1e2) and exons 3 and 8 (for IGF2 -P1e3) were used. This approach was necessary because specific amplification of transcripts derived from P1 promoter failed due to lack of suitable PCR primer sequence in exon 1. Since IGF2 -transcript P1 exon 2 is part of the first exonic region of transcript IGF2 -P0, and exon 3 is present in both IGF2- P0 and IGF2- P1 transcripts, the IGF2 -P1e2 and -P1e3 amplicons could potentially derive from P0 and/or P1 promoters, depending on tissue and developmental stage. We quantified transcript abundances for two splice variants derived from IGF2- P2 promoter which comprise leading exon 4 ( IGF2 -P2e4) or leading exons 4 and 5 ( IGF2 -P2e5) as well as the protein coding exons. The forward primer for IGF2 -P2e4 was designed to span the junction of exons 4 and 8, and for IGF2 -P2e5 was in exon 5 with the reverse primer for both splice variants in exon 8. To amplify IGF2 -P3 and IGF2 -P4 transcripts, forward primers were designed within exons 6 and 7 with the reverse primer located within exon 8. All primers are detailed in S3 Table .

    Article Snippet: Target specificity and integrity was confirmed via sequencing of selected amplicons on a 3730xl DNA Analyzer (Applied Biosystems, Inc., Foster City, CA, USA), plots of the melting curve derived by Mastercycler® ep Realplex software (Eppendorf, Inc., Hamburg, Germany), and by electrophoresis of PCR products on a 2% agarose gel (Agarose low EEO, AppliChem GmbH, Darmstadt, Germany) and visual inspection under UV with Gel DocTM 1000 Single Wavelength Mini-Transilluminator, using Quantity One image analyzing software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) after staining with GelRed™ Nucleic Acid Stain (Biotium, Inc., Hayward, CA, USA).

    Techniques: Amplification, Variant Assay, Sequencing, Derivative Assay, Polymerase Chain Reaction

    Autophagy enhances NFκB activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB p65 (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in DNA was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p

    Journal: Nature communications

    Article Title: Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity

    doi: 10.1038/ncomms6779

    Figure Lengend Snippet: Autophagy enhances NFκB activation in F4/80 hi macrophages (a) Production of CXCL1 and CXCL2 by F4/80 lo and F4/80 hi peritoneal macrophages. Cells were stimulated with zymosan (100 μg/ml) or (HKCA; 5×10 5 spores/ml) for 24 hrs with or without an NFκB inhibitor, QNZ. Culture supernatants were analyzed by ELISA. n=3. ( b ) Western blotting analysis of p-IκB and total IκB in F4/80 hi peritoneal macrophages stimulated with zymosan (100 μg/ml) for 30 min. ( c ) Translocation of NFκB p65 (green) to the nucleus (DAPI, blue) in F4/80 hi peritoneal macrophages stimulated with Pam 3 CSK 4 (100 ng/ml) for 30 min. NFκB localization was analyzed by confocal microscopy. Yellow broken lines in enlarged photos indicate borders of the nucleus. Shown are representative images from at least 50 cells. Scale bars indicate 5 μm. ( d ) Relative activity of NFκB in WT and Atg7 CKO F4/80 hi peritoneal macrophages was evaluated. Cells were stimulated with zymosan (100 μg/ml) for 30 min, and p62 binding to a target NF-κB response element in DNA was evaluated by colorimetric reading. Values were calculated as a fold increase of O.D. values of stimulated cells from those of unstimulated cells. n=3. Data are representative of two independent experiments. Error bars represent mean ± SD. ANOVA and Student’s t -test were used for statistical analysis of (a) and (d), respectively. N.S.; not significant. ***; p

    Article Snippet: Second, functional NFκB activity was carried out by evaluating the binding of p65 to a target DNA fragment wtih NFκB p65 Transcription Factor Assay kit (Thermo scientific).

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Translocation Assay, Confocal Microscopy, Activity Assay, Binding Assay