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Proteintech anti e1b ap5
The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, <t>E1B-AP5,</t> and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
Anti E1b Ap5, supplied by Proteintech, used in various techniques. Bioz Stars score: 85/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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85/100 stars

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1) Product Images from "Influenza virus targets the mRNA export machinery and the nuclear pore complex"

Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex

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

doi: 10.1073/pnas.0610977104

The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
Figure Legend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

Techniques Used: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling

2) Product Images from "Influenza virus targets the mRNA export machinery and the nuclear pore complex"

Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex

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

doi: 10.1073/pnas.0610977104

The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
Figure Legend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

Techniques Used: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling

Selective impairment of mRNA nuclear export in Rae1 and Nup98 mutant cells. Nuclear (N) and cytoplasmic (C) RNA was isolated from Rae1 +/+ Nup98 +/+ , Rae1 +/+ Nup98 +/− , Rae1 +/− Nup98 +/+ , and Rae1 +/− Nup98 +/− MEFs. Relative mRNA levels were quantified by real-time RT-PCR using gene-specific primers and normalized to a set of five housekeeping genes.
Figure Legend Snippet: Selective impairment of mRNA nuclear export in Rae1 and Nup98 mutant cells. Nuclear (N) and cytoplasmic (C) RNA was isolated from Rae1 +/+ Nup98 +/+ , Rae1 +/+ Nup98 +/− , Rae1 +/− Nup98 +/+ , and Rae1 +/− Nup98 +/− MEFs. Relative mRNA levels were quantified by real-time RT-PCR using gene-specific primers and normalized to a set of five housekeeping genes.

Techniques Used: Mutagenesis, Isolation, Quantitative RT-PCR

Low levels of Rae1 and Nup98 induce higher susceptibility to influenza virus-mediated cell death and an increase in viral replication. ( a and b ) Rae1 +/+ Nup98 +/+ , Rae1 +/− Nup98 +/+ , Rae1 +/+ Nup98 +/− , and Rae1 +/− Nup98 +/− MEFs were infected with A/WS/33 influenza virus, and cell viability was determined by comparing and quantifying bright-field microscopy (gray), DAPI (blue), and exclusion of 2 mM ethidium homodimer-1 (red). ( c ) The number of influenza viral particles was measured in the supernatants of the cells in a by using the hemaglutinin assay.
Figure Legend Snippet: Low levels of Rae1 and Nup98 induce higher susceptibility to influenza virus-mediated cell death and an increase in viral replication. ( a and b ) Rae1 +/+ Nup98 +/+ , Rae1 +/− Nup98 +/+ , Rae1 +/+ Nup98 +/− , and Rae1 +/− Nup98 +/− MEFs were infected with A/WS/33 influenza virus, and cell viability was determined by comparing and quantifying bright-field microscopy (gray), DAPI (blue), and exclusion of 2 mM ethidium homodimer-1 (red). ( c ) The number of influenza viral particles was measured in the supernatants of the cells in a by using the hemaglutinin assay.

Techniques Used: Infection, Microscopy

The mRNA export inhibition induced by NS1 is reverted by increased levels of mRNA export factors. ( a and b ) Luciferase reporter gene expression assays were performed with 293T cells by cotransfection of reporter plasmids and plasmids encoding NXF1, p15, Rae1, Nup98, and Nup96, as indicated. ( c ) HeLa cells were transfected with a plasmid encoding myc-NS1 alone or cotransfected with plasmids encoding myc-NS1, GFP-NXF1, and GFP-p15. Cells were subjected to immunofluorescence with anti-myc antibody (red) followed by oligo(dT) in situ hybridization (blue). Green shows GFP-NXF1 and GFP-p15.
Figure Legend Snippet: The mRNA export inhibition induced by NS1 is reverted by increased levels of mRNA export factors. ( a and b ) Luciferase reporter gene expression assays were performed with 293T cells by cotransfection of reporter plasmids and plasmids encoding NXF1, p15, Rae1, Nup98, and Nup96, as indicated. ( c ) HeLa cells were transfected with a plasmid encoding myc-NS1 alone or cotransfected with plasmids encoding myc-NS1, GFP-NXF1, and GFP-p15. Cells were subjected to immunofluorescence with anti-myc antibody (red) followed by oligo(dT) in situ hybridization (blue). Green shows GFP-NXF1 and GFP-p15.

Techniques Used: Inhibition, Luciferase, Expressing, Cotransfection, Transfection, Plasmid Preparation, Immunofluorescence, In Situ Hybridization

3) Product Images from "Influenza virus targets the mRNA export machinery and the nuclear pore complex"

Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex

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

doi: 10.1073/pnas.0610977104

The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
Figure Legend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

Techniques Used: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling

Influenza virus inhibits poly(A) RNA nuclear export. ( a ) MDCK cells were mock infected or infected with A/WS/33 influenza virus at an MOI of 1 for 6 and 24 h. Immunofluorescence using antibodies against influenza proteins (green) and oligo(dT) in situ hybridization (red) was performed. ( b ) Expression of influenza proteins in MDCKs. Cell extracts from MDCK cells infected with A/WS/33 at an MOI 1 for the indicated time points were subjected to immunoblot analysis with anti-influenza protein antibodies.
Figure Legend Snippet: Influenza virus inhibits poly(A) RNA nuclear export. ( a ) MDCK cells were mock infected or infected with A/WS/33 influenza virus at an MOI of 1 for 6 and 24 h. Immunofluorescence using antibodies against influenza proteins (green) and oligo(dT) in situ hybridization (red) was performed. ( b ) Expression of influenza proteins in MDCKs. Cell extracts from MDCK cells infected with A/WS/33 at an MOI 1 for the indicated time points were subjected to immunoblot analysis with anti-influenza protein antibodies.

Techniques Used: Infection, Immunofluorescence, In Situ Hybridization, Expressing

4) Product Images from "Stability of a Long Noncoding Viral RNA Depends on a 9-nt Core Element at the RNA 5' End to Interact with Viral ORF57 and Cellular PABPC1"

Article Title: Stability of a Long Noncoding Viral RNA Depends on a 9-nt Core Element at the RNA 5' End to Interact with Viral ORF57 and Cellular PABPC1

Journal: International Journal of Biological Sciences

doi:

MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. ( A ) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4 A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells 39 induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA 27 which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. ( B ) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and E1B-AP5 antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA 27 were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4 B. Control beads indicate no RNA oligomer on the beads.
Figure Legend Snippet: MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. ( A ) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4 A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells 39 induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA 27 which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. ( B ) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and E1B-AP5 antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA 27 were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4 B. Control beads indicate no RNA oligomer on the beads.

Techniques Used: In Vitro, Binding Assay, Sequencing, Derivative Assay, Positive Control, Western Blot, Plasmid Preparation

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    Proteintech anti e1b ap5
    The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, <t>E1B-AP5,</t> and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .
    Anti E1b Ap5, supplied by Proteintech, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti e1b ap5/product/Proteintech
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti e1b ap5 - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    99
    Proteintech rabbit polyclonal anti e1b ap5
    MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. ( A ) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4 A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells 39 induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA 27 which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. ( B ) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and <t>E1B-AP5</t> antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA 27 were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4 B. Control beads indicate no RNA oligomer on the beads.
    Rabbit Polyclonal Anti E1b Ap5, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti e1b ap5/product/Proteintech
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti e1b ap5 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

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

    Article Title: Influenza virus targets the mRNA export machinery and the nuclear pore complex

    doi: 10.1073/pnas.0610977104

    Figure Lengend Snippet: The NS1 protein of influenza virus interacts with key constituents of the mRNA export pathway. ( a ) Cell lysates from 293T cells were incubated with immobilized recombinant GST or GST-NS1. Bound and unbound (UB) fractions were analyzed by 4–20% SDS/PAGE followed by immunoblot analysis with antibodies to NXF1, p15, Rae1, E1B-AP5, and Nup98. ( b and c ) Experiments were performed as in a except that antibodies against Nup96 and against Nup62 and Nup153 (mAb414) were used for immunoblot analysis. ( d ) GST-NS1 or the amino-terminal or carboxyl-terminal domains of NS1 fused with GST were incubated with cell lysates and processed as in a . ( e ) GST-NS1 was incubated with cell lysates untreated or treated with RNase A and processed as in a . ( f and g ) Expression levels of Nups and mRNA export factors in 293T ( f ) and MDCK ( g ) cells infected with influenza virus. Cell extracts were subjected to immunoblot analysis with antibodies against Nup98, β-actin, Rae1, NXF1, and E1B-AP5, and with mAb414 antibodies. ( h ) Half-life measurements of Nup98. MDCK cells were pulse-labeled for 2 h and chased for the depicted time points. Immunoprecipitations were performed with anti-Nup98 antibodies or preimmune serum (PI). Nup98 bands were analyzed by densitometry as described in Methods .

    Article Snippet: Immunoblot analysis was performed with anti-Nup98 , anti-E1B-AP5 (Proteintech Group, Chicago, IL), anti-Rae1 , and anti-NXF1/TAP antibodies (BD Transduction Laboratories, San Jose, CA), anti-p15/NXT antibodies (Abnova, Taipei City, Taiwan), mAb414 , and anti-Influenza A antibodies (virions; Biodesign International, Saco, ME).

    Techniques: Incubation, Recombinant, SDS Page, Expressing, Infection, Labeling

    MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. ( A ) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4 A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells 39 induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA 27 which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. ( B ) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and E1B-AP5 antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA 27 were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4 B. Control beads indicate no RNA oligomer on the beads.

    Journal: International Journal of Biological Sciences

    Article Title: Stability of a Long Noncoding Viral RNA Depends on a 9-nt Core Element at the RNA 5' End to Interact with Viral ORF57 and Cellular PABPC1

    doi:

    Figure Lengend Snippet: MRE-II RNA interacts in vitro with ORF57 and other cellular proteins. ( A ) Mapping of an MRE binding site for ORF57. Shown on the top is a PAN MRE sequence lacking the MRE-I region in Fig. 4 A, with the 9-nt core of MRE-II loop sequence bolded and underlined and the nt positions of biotinylated RNA oligomers employed in RNA pull-down assays. A cell lysate prepared from TREx BCBL-1-Rta cells 39 induced with Dox for 24 h was used for the RNA pulldown assays with an indicated RNA oligomer. The RNA oligo oNP42 derived from vIL-6 RNA 27 which binds ORF57 was used as a positive control. ORF57 associated with RNA oligos in the pulldowns was immunoblotted using an anti-ORF57 antibody. The cell lysate (10%) before the pulldown was loaded as a Western blot control. ( B ) Biotinylated RNA affinity pulldown analysis by Western blot using anti-ORF57, PABPC1 and E1B-AP5 antibodies. Total cell extract from TREx BCBL-1-Rta (R) or -vector (V) cells induced by Dox for 24 h was used for the RNA pulldown assays with each biotinylated RNA oligomer. RNA oligos oNP41 and oNP42 derived from vIL-6 RNA 27 were used, respectively, as a negative and positive oligomer control for ORF57 interaction and 10% of the cell lysate from each cell line before the pulldown was loaded for Western blotting control. RNA oligo oJM68 is a copy of oJM35 with the point mutations in the MRE-II loop described in Fig. 4 B. Control beads indicate no RNA oligomer on the beads.

    Article Snippet: The following antibodies were used in Western blot analyses: a rabbit polyclonal anti-ORF57 antibody (1:3,000 dilution), mouse monoclonal anti-ORF57 antibody (unpublished data, used at a dilution of 1:1,000), rabbit polyclonal anti-PABPC1 (1:700, Abcam ab21060), and rabbit polyclonal anti-E1B-AP5 (1:1,000, ProteinTech Group 0578-1-AP, Chicago, IL), together with corresponding peroxidase-conjugated secondary antibodies (1:10,000, Sigma).

    Techniques: In Vitro, Binding Assay, Sequencing, Derivative Assay, Positive Control, Western Blot, Plasmid Preparation