Structured Review

Promega human β globin gene
Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human <t>β-globin</t> intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
Human β Globin Gene, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human β globin gene/product/Promega
Average 89 stars, based on 2 article reviews
Price from $9.99 to $1999.99
human β globin gene - by Bioz Stars, 2020-07
89/100 stars

Images

1) Product Images from "Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2"

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp497

Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
Figure Legend Snippet: Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

Techniques Used: Luciferase, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Purification, Activity Assay, Quantitative RT-PCR

2) Product Images from "Specific repression of ?-globin promoter activity by nuclear ferritin"

Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin

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

doi: 10.1073/pnas.151147098

Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.
Figure Legend Snippet: Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.

Techniques Used: Binding Assay, Labeling, Purification

Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .
Figure Legend Snippet: Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .

Techniques Used: Cotransfection, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .
Figure Legend Snippet: Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .

Techniques Used: Binding Assay

Related Articles

Clone Assay:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: .. For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega). .. The expression plasmid for UL69 (pCMV-UL69) was kindly provided by T. Stamminger ( ).

Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin
Article Snippet: .. The upstream region (−610/+20) of the human β-globin gene, previously cloned in pSV2CAT , was subcloned through pGEM and pSELECT (now called pALTER) and recloned into pCAT-basic (all vectors from Promega). .. Mutants of the −153/−148 promoter site were generated by transcription from mutant oligonucleotides corresponding to the −164/−128 region by using the pSELECT system.

Amplification:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: .. For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega). .. The expression plasmid for UL69 (pCMV-UL69) was kindly provided by T. Stamminger ( ).

Polymerase Chain Reaction:

Article Title: Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches
Article Snippet: .. The artificial intron (133 bp), composed of the 5′ donor splice site from the first intron of the human β-globin gene and the branch point and 3′ acceptor splice site (3′SS) from the intron of an immunoglobulin gene heavy chain variable region, was subcloned by PCR from pCI-neo Mammalian Expression Vector (Promega) into Ale1 blunt restriction site. pSMD2-ΔCMV-AS2-3′SSC-E23-E59/79opt and pSMD2-Δlinker-E23-E59/79opt were created from pSMD2-AS2-3′SSC-E23-E59/79opt by PCR, deleting, respectively, the CMV promoter and the hemi-intron. .. All expression cassettes subcloned in pSMD2 backbone are under the control of the strong CMV promoter and a polyA signal.

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: .. For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega). .. The expression plasmid for UL69 (pCMV-UL69) was kindly provided by T. Stamminger ( ).

Expressing:

Article Title: Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches
Article Snippet: .. The artificial intron (133 bp), composed of the 5′ donor splice site from the first intron of the human β-globin gene and the branch point and 3′ acceptor splice site (3′SS) from the intron of an immunoglobulin gene heavy chain variable region, was subcloned by PCR from pCI-neo Mammalian Expression Vector (Promega) into Ale1 blunt restriction site. pSMD2-ΔCMV-AS2-3′SSC-E23-E59/79opt and pSMD2-Δlinker-E23-E59/79opt were created from pSMD2-AS2-3′SSC-E23-E59/79opt by PCR, deleting, respectively, the CMV promoter and the hemi-intron. .. All expression cassettes subcloned in pSMD2 backbone are under the control of the strong CMV promoter and a polyA signal.

Sequencing:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: .. For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega). .. The expression plasmid for UL69 (pCMV-UL69) was kindly provided by T. Stamminger ( ).

Plasmid Preparation:

Article Title: Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches
Article Snippet: .. The artificial intron (133 bp), composed of the 5′ donor splice site from the first intron of the human β-globin gene and the branch point and 3′ acceptor splice site (3′SS) from the intron of an immunoglobulin gene heavy chain variable region, was subcloned by PCR from pCI-neo Mammalian Expression Vector (Promega) into Ale1 blunt restriction site. pSMD2-ΔCMV-AS2-3′SSC-E23-E59/79opt and pSMD2-Δlinker-E23-E59/79opt were created from pSMD2-AS2-3′SSC-E23-E59/79opt by PCR, deleting, respectively, the CMV promoter and the hemi-intron. .. All expression cassettes subcloned in pSMD2 backbone are under the control of the strong CMV promoter and a polyA signal.

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: .. For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega). .. The expression plasmid for UL69 (pCMV-UL69) was kindly provided by T. Stamminger ( ).

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  • 89
    Promega human β globin gene
    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human <t>β-globin</t> intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
    Human β Globin Gene, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human β globin gene/product/Promega
    Average 89 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    human β globin gene - by Bioz Stars, 2020-07
    89/100 stars
      Buy from Supplier

    88
    Promega human beta globin gene
    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human <t>β-globin</t> intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
    Human Beta Globin Gene, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human beta globin gene/product/Promega
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human beta globin gene - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    99
    Promega β globin
    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human <t>β-globin</t> intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.
    β Globin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β globin/product/Promega
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    β globin - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

    Journal: Nucleic Acids Research

    Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2

    doi: 10.1093/nar/gkp497

    Figure Lengend Snippet: Translation stimulation does not occur with spliced mRNAs. ( A ) Schematic representation of the intronless (pcDNAGlobinRen) and the intron-containing (pcDNAIntronGlobinRen) vectors encoding the Renilla luciferase showing positions of the human β-globin intron within the 5′UTR of the luciferase construct (gray arrows correspond to positions of the PCR primers used to test efficient splicing of the intron). ( B ) RT-PCR (using primers shown in A) from cells cotransfected with pcDNAGlobinRen and pCI or the EB2-encoding plasmid pCI-FlagEB2 (lanes 2 and 3) or from cells cotransfected with pcDNAIntronGlobinRen and pCI or pCI-FlagEB2 (lanes 5 and 6), or directly from the purified DNA vector (lanes 4 and 7). ( C ) Luciferase activity normalized by reference to the amount of cytoplasmic luciferase-encoding mRNAs from HeLa cells cotransfected with pcDNAGlobinRen and pCI or pCI-FlagEB2, or pcDNAIntronGlobinRen and pCI or pCI-FlagEB2. The amount of luciferase-encoding mRNAs was monitored by quantitative RT-PCR.

    Article Snippet: For the pcDNAIntron-GlobinRen, the sequence corresponding to the intron of the human β-globin gene was amplified by PCR and cloned into the pcDNAGlobinRen vector previously digested by XbaI. pCI-mycORF57 contained the complete ORF57 coding sequence (first exon included) cloned in frame with the myc epitope, in pCI (Promega).

    Techniques: Luciferase, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Purification, Activity Assay, Quantitative RT-PCR

    Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.

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

    Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin

    doi: 10.1073/pnas.151147098

    Figure Lengend Snippet: Binding of ferritin chains from human heart and liver to the distal promoter of the human β-globin gene. The Rsa fragment of the β-globin promoter (−222/−128) was end-labeled with 32 ), with 1 μl of K562 nuclear extract (lane 2) or 2–4 ng of purified proteins, as follows: 2 ng of human liver ferritin (lane 3), 4 ng of human heart ferritin (lane 4), 2 ng each of human transferrin (lane 5) and apotransferrin (lane 6). Shift bands are denoted by arrowheads at the left. Lane 1 contained only DNA. A second fragment of the β-globin promoter (−127/+20) gave no shift bands with either 2 ng (lane 7) or 4 ng (lane 8) of liver ferritin or 4 ng of heart ferritin (lane 9). Only ferritin enriched in H-chains gave a strong shift (lane 4). The DNA-binding reactions for this figure were carried out at 37°C.

    Article Snippet: The upstream region (−610/+20) of the human β-globin gene, previously cloned in pSV2CAT , was subcloned through pGEM and pSELECT (now called pALTER) and recloned into pCAT-basic (all vectors from Promega).

    Techniques: Binding Assay, Labeling, Purification

    Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .

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

    Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin

    doi: 10.1073/pnas.151147098

    Figure Lengend Snippet: Cotransfection experiments demonstrating ferritin-H repression of the β-globin promoter and loss of ability to repress when the ferritin binding site (CAGTGC) is mutated. Transfections of CV-1 cells were performed with a constant amount (6 μg) of total plasmid DNAs mixed with 8 μl of DMRIE-C added to 2 × 10 6 CV-1 cells, such that each transfection had 2 μg of β-CAT plasmid (W = wt, or M = mutant), ±1 μg of EKLF, ±3 μg of F H (ferritin-H expression plasmid), with the difference made up to 6 μg with pEGFP. Reporter gene activity, expressed as ng of CAT per mg of cellular protein (measured by ELISA), is shown for the following combinations with either native (W) or mutant (M) β-CAT plasmids: the nonstimulated human β-globin promoter (open bars); the β-globin promoter stimulated by a cotransfected EKLF effector plasmid (hatched bars); and EKLF-stimulated β-globin promoter cotransfected with a ferritin-H expression plasmid (solid bars). ( n = 3 transfections per data set; bars = SEM). Construction of reporter plasmids (diagrammed above the histogram) is described in Materials and Methods .

    Article Snippet: The upstream region (−610/+20) of the human β-globin gene, previously cloned in pSV2CAT , was subcloned through pGEM and pSELECT (now called pALTER) and recloned into pCAT-basic (all vectors from Promega).

    Techniques: Cotransfection, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

    Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .

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

    Article Title: Specific repression of ?-globin promoter activity by nuclear ferritin

    doi: 10.1073/pnas.151147098

    Figure Lengend Snippet: Nuclear anti-ferritin-reactive protein binds the β-globin promoter. ( a ) Diagram of the human β-globin locus, a 5′ region of the β-globin gene (−250 to +20), and specific segments used in b , i.e., the distal promoter (−222/−128) and two double-stranded oligonucleotides, −164/−128 and −232/−188. ( b ) Binding of a ferritin-family protein from K562 cell nuclear extracts to the −222/−128 β-globin region, and localization of the binding to the −164/−128 segment by using an antibody supershift assay described under Materials and Methods .

    Article Snippet: The upstream region (−610/+20) of the human β-globin gene, previously cloned in pSV2CAT , was subcloned through pGEM and pSELECT (now called pALTER) and recloned into pCAT-basic (all vectors from Promega).

    Techniques: Binding Assay