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    Thermo Fisher anti nup50
    Interaction of nuclear transport receptors with <t>Nup50.</t> (A) NRK cells were solubilized in NP-40 buffer, and extracts were immunoprecipitated with anti-Nup50 antibodies (antibody 1). Material bound to the antibody beads (b) and the unbound material (ub) were electrophoresed on an SDS gel and analyzed by immunoblotting with anti-CRM1, anti-CAS, anti-importin β, or anti-lamin antibodies. Note that the sample loaded in the bound lanes represents sevenfold more cell equivalents than the material in the unbound lanes. (B) Sepharose beads coupled with a Nup50 fragment (Nup50) or with BSA were incubated with recombinant CRM1, CAS, or importin β in the absence or presence of RanGMP-PNP or cytochrome c coupled with NES peptides (cc-NES) as indicated. Samples were analyzed by immunoblotting with antibodies recognizing the His 6 tag of the recombinant receptors. An equivalent amount of the bound material was loaded in all cases. Moreover, the input material contained nearly equal quantities of all transport receptors, as determined by protein analysis. (C) NRK cells were incubated at 37°C for 4 h in growth medium containing 100 nM leptomycin B (+LMB) or lacking leptomycin B (−LMB). They were then fixed with methanol-acetone (see Materials and Methods) and examined by confocal microscopy after double immunofluorescence staining with anti-Nup50 (antibody 1) or RL1, as indicated.
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    Interaction of nuclear transport receptors with Nup50. (A) NRK cells were solubilized in NP-40 buffer, and extracts were immunoprecipitated with anti-Nup50 antibodies (antibody 1). Material bound to the antibody beads (b) and the unbound material (ub) were electrophoresed on an SDS gel and analyzed by immunoblotting with anti-CRM1, anti-CAS, anti-importin β, or anti-lamin antibodies. Note that the sample loaded in the bound lanes represents sevenfold more cell equivalents than the material in the unbound lanes. (B) Sepharose beads coupled with a Nup50 fragment (Nup50) or with BSA were incubated with recombinant CRM1, CAS, or importin β in the absence or presence of RanGMP-PNP or cytochrome c coupled with NES peptides (cc-NES) as indicated. Samples were analyzed by immunoblotting with antibodies recognizing the His 6 tag of the recombinant receptors. An equivalent amount of the bound material was loaded in all cases. Moreover, the input material contained nearly equal quantities of all transport receptors, as determined by protein analysis. (C) NRK cells were incubated at 37°C for 4 h in growth medium containing 100 nM leptomycin B (+LMB) or lacking leptomycin B (−LMB). They were then fixed with methanol-acetone (see Materials and Methods) and examined by confocal microscopy after double immunofluorescence staining with anti-Nup50 (antibody 1) or RL1, as indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Interaction of nuclear transport receptors with Nup50. (A) NRK cells were solubilized in NP-40 buffer, and extracts were immunoprecipitated with anti-Nup50 antibodies (antibody 1). Material bound to the antibody beads (b) and the unbound material (ub) were electrophoresed on an SDS gel and analyzed by immunoblotting with anti-CRM1, anti-CAS, anti-importin β, or anti-lamin antibodies. Note that the sample loaded in the bound lanes represents sevenfold more cell equivalents than the material in the unbound lanes. (B) Sepharose beads coupled with a Nup50 fragment (Nup50) or with BSA were incubated with recombinant CRM1, CAS, or importin β in the absence or presence of RanGMP-PNP or cytochrome c coupled with NES peptides (cc-NES) as indicated. Samples were analyzed by immunoblotting with antibodies recognizing the His 6 tag of the recombinant receptors. An equivalent amount of the bound material was loaded in all cases. Moreover, the input material contained nearly equal quantities of all transport receptors, as determined by protein analysis. (C) NRK cells were incubated at 37°C for 4 h in growth medium containing 100 nM leptomycin B (+LMB) or lacking leptomycin B (−LMB). They were then fixed with methanol-acetone (see Materials and Methods) and examined by confocal microscopy after double immunofluorescence staining with anti-Nup50 (antibody 1) or RL1, as indicated.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Immunoprecipitation, SDS-Gel, Incubation, Recombinant, Confocal Microscopy, Double Immunofluorescence Staining

    Analysis of nuclear protein export in NRK cells injected with anti-Nup50 antibodies. The nuclei of cells were injected with a marker 100-kDa fluorescent dextran, together with GST-NES mixed with buffer (A) or with GST-NES mixed with antibody 1 (B), antibody 2 (C), or antibody 1 plus immunizing antigen (D), as shown. After 30 min at 37°C, cells were fixed, and the localization of GST-NES was determined by indirect immunofluorescence microscopy. Injected nuclei are denoted by the fluorescent dextran.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Analysis of nuclear protein export in NRK cells injected with anti-Nup50 antibodies. The nuclei of cells were injected with a marker 100-kDa fluorescent dextran, together with GST-NES mixed with buffer (A) or with GST-NES mixed with antibody 1 (B), antibody 2 (C), or antibody 1 plus immunizing antigen (D), as shown. After 30 min at 37°C, cells were fixed, and the localization of GST-NES was determined by indirect immunofluorescence microscopy. Injected nuclei are denoted by the fluorescent dextran.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Injection, Marker, Immunofluorescence, Microscopy

    Immunoblot analysis of Nup50 antigens in various cell lines and tissues from rats. Samples of various rat tissues (A) or cultured cell lines (B), as indicated, were analyzed by immunoblotting with antibody 1 (panel A and the left part part of panel B) and antibody 2 (the right part of panel B). The positions of Nup50 and the major ∼70-kDa antigen that is recognized by antibody 1 are indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Immunoblot analysis of Nup50 antigens in various cell lines and tissues from rats. Samples of various rat tissues (A) or cultured cell lines (B), as indicated, were analyzed by immunoblotting with antibody 1 (panel A and the left part part of panel B) and antibody 2 (the right part of panel B). The positions of Nup50 and the major ∼70-kDa antigen that is recognized by antibody 1 are indicated.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Cell Culture

    MALDI mass spectrometry analysis of Nup50 and p70 antigens of NRK cells. Gel-purified Nup50 and p70 bands from anti-Nup50 immunoprecipitates of NRK cells were digested with trypsin, and the products were analyzed by MALDI time-of-flight mass spectrometry. Short bars indicate peptides smaller than 3,000 Da from each band that match (within experimental accuracy) the predicted mass of peptides from the deduced cDNA sequence of rat Nup50. The total regions of the predicted sequence of Nup50 that are represented by peptides obtained in the MALDI analysis are indicated by filled regions of the top bar designating the linear sequence of Nup50.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: MALDI mass spectrometry analysis of Nup50 and p70 antigens of NRK cells. Gel-purified Nup50 and p70 bands from anti-Nup50 immunoprecipitates of NRK cells were digested with trypsin, and the products were analyzed by MALDI time-of-flight mass spectrometry. Short bars indicate peptides smaller than 3,000 Da from each band that match (within experimental accuracy) the predicted mass of peptides from the deduced cDNA sequence of rat Nup50. The total regions of the predicted sequence of Nup50 that are represented by peptides obtained in the MALDI analysis are indicated by filled regions of the top bar designating the linear sequence of Nup50.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Mass Spectrometry, Purification, Sequencing

    Immunofluorescent staining of rat liver nuclei and NRK cells with anti-Nup50 antibodies. Rat liver cryosections (A), isolated rat liver nuclei (B), or NRK cells (C and D) were fixed with either formaldehyde (F panels) or methanol-acetone (M panels) and labeled with antibody 1 (A to C) or antibody 2 (D) against Nup50 for visualization by indirect immunofluorescence microscopy.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Immunofluorescent staining of rat liver nuclei and NRK cells with anti-Nup50 antibodies. Rat liver cryosections (A), isolated rat liver nuclei (B), or NRK cells (C and D) were fixed with either formaldehyde (F panels) or methanol-acetone (M panels) and labeled with antibody 1 (A to C) or antibody 2 (D) against Nup50 for visualization by indirect immunofluorescence microscopy.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Staining, Isolation, Labeling, Immunofluorescence, Microscopy

    Distribution of Nup50 by subcellular fractionation. (A) Isolated rat liver nuclei (Nuc) were digested with DNase-RNase to release intranuclear contents from the NE and were centrifuged at low speed to yield a supernatant (Sup) and pellet (Pel). (B) Isolated rat liver NE, derived by nuclease-salt extraction of nuclei, were suspended in a solution containing 0.5 M NaCl and were centrifuged to yield a supernatant and a pellet. (C) NRK cells (Cell) were permeabilized, digested with DNase-RNase, suspended in buffer containing 0.5 M NaCl, and centrifuged to yield a supernatant and pellet. (D) Rat liver NEs were solubilized in nonionic detergent buffer and were passed over a WGA affinity column, yielding bound and unbound fractions. Samples of the various subcellular fractions were electrophoresed on SDS gels and analyzed by immunoblotting with antibody 1 or the monoclonal antibody RL1 as indicated.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Distribution of Nup50 by subcellular fractionation. (A) Isolated rat liver nuclei (Nuc) were digested with DNase-RNase to release intranuclear contents from the NE and were centrifuged at low speed to yield a supernatant (Sup) and pellet (Pel). (B) Isolated rat liver NE, derived by nuclease-salt extraction of nuclei, were suspended in a solution containing 0.5 M NaCl and were centrifuged to yield a supernatant and a pellet. (C) NRK cells (Cell) were permeabilized, digested with DNase-RNase, suspended in buffer containing 0.5 M NaCl, and centrifuged to yield a supernatant and pellet. (D) Rat liver NEs were solubilized in nonionic detergent buffer and were passed over a WGA affinity column, yielding bound and unbound fractions. Samples of the various subcellular fractions were electrophoresed on SDS gels and analyzed by immunoblotting with antibody 1 or the monoclonal antibody RL1 as indicated.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Fractionation, Isolation, Derivative Assay, Whole Genome Amplification, Affinity Column

    Analysis of nuclear protein import in NRK cells injected with anti-Nup50 antibodies. (A) Cells were injected in the cytoplasm with GST-NLS. After either 5 or 30 min at 37°C, cells were fixed and the GST-NLS was localized by indirect immunofluorescence microscopy. (B) Cells were injected into the nucleus with a mixture of a marker (100-kDa fluorescent dextran) and antibody 1. They were then injected into the cytoplasm with GST-NLS. After either 5 or 30 min at 37°C, cells were fixed and the GST-NLS was localized by indirect immunofluorescence microscopy. Injected nuclei are denoted by the fluorescent dextran.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Analysis of nuclear protein import in NRK cells injected with anti-Nup50 antibodies. (A) Cells were injected in the cytoplasm with GST-NLS. After either 5 or 30 min at 37°C, cells were fixed and the GST-NLS was localized by indirect immunofluorescence microscopy. (B) Cells were injected into the nucleus with a mixture of a marker (100-kDa fluorescent dextran) and antibody 1. They were then injected into the cytoplasm with GST-NLS. After either 5 or 30 min at 37°C, cells were fixed and the GST-NLS was localized by indirect immunofluorescence microscopy. Injected nuclei are denoted by the fluorescent dextran.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Injection, Immunofluorescence, Microscopy, Marker

    Immunoelectron microscopic localization of Nup50 in NEs and NRK cells. (A) Isolated rat liver NEs were labeled by indirect immunogold techniques with anti-Nup50 antibodies (antibody 1). Shown are cross-sectional (left two panels) and tangential (right panels) views of the NE depicting the labeling pattern that was obtained. Undigested chromatin associated with the inner nuclear membrane (Ch) allows distinction between the cytoplasmic and nucleoplasmic sides of the NPC. Arrowheads denote NPCs that are labeled with anti-Nup50 antibodies. (B) Isolated rat liver NEs were processed by indirect immunogold labeling to simultaneously localize Nup50 (antibody 1, 10-nm gold, arrow in left panel) and Nup153 (RL11, 5-nm gold, arrowhead in left panel). (C) NRK cells were permeabilized by freeze-thawing, and Nup50 was localized by indirect immunogold labeling. The top row of panels depicts cross-sectional views of the NE, which reveal gold labeling exclusively on the nucleoplasmic side of the NPC. The bottom panel presents a cross-section through the nucleus, showing that gold labeling is present in more internal nuclear regions (arrowheads), as well as at the nucleoplasmic surface of the NPC. Bars, 100 nm. (D) Quantification of the localization of Nup50 and Nup153 in isolated rat liver NEs with respect to the NPC midplane. One hundred eighteen gold particles were counted for Nup50, and 110 were counted for Nup153. (E) Quantification of the localization of Nup50 at the NE of NRK cells with respect to the NPC midplane. Eighty-four gold particles were counted.

    Journal: Molecular and Cellular Biology

    Article Title: Nup50, a Nucleoplasmically Oriented Nucleoporin with a Role in Nuclear Protein Export

    doi:

    Figure Lengend Snippet: Immunoelectron microscopic localization of Nup50 in NEs and NRK cells. (A) Isolated rat liver NEs were labeled by indirect immunogold techniques with anti-Nup50 antibodies (antibody 1). Shown are cross-sectional (left two panels) and tangential (right panels) views of the NE depicting the labeling pattern that was obtained. Undigested chromatin associated with the inner nuclear membrane (Ch) allows distinction between the cytoplasmic and nucleoplasmic sides of the NPC. Arrowheads denote NPCs that are labeled with anti-Nup50 antibodies. (B) Isolated rat liver NEs were processed by indirect immunogold labeling to simultaneously localize Nup50 (antibody 1, 10-nm gold, arrow in left panel) and Nup153 (RL11, 5-nm gold, arrowhead in left panel). (C) NRK cells were permeabilized by freeze-thawing, and Nup50 was localized by indirect immunogold labeling. The top row of panels depicts cross-sectional views of the NE, which reveal gold labeling exclusively on the nucleoplasmic side of the NPC. The bottom panel presents a cross-section through the nucleus, showing that gold labeling is present in more internal nuclear regions (arrowheads), as well as at the nucleoplasmic surface of the NPC. Bars, 100 nm. (D) Quantification of the localization of Nup50 and Nup153 in isolated rat liver NEs with respect to the NPC midplane. One hundred eighteen gold particles were counted for Nup50, and 110 were counted for Nup153. (E) Quantification of the localization of Nup50 at the NE of NRK cells with respect to the NPC midplane. Eighty-four gold particles were counted.

    Article Snippet: After fixation and permeabilization, the coverslips were incubated for 30 min at RT in PBS containing 0.1% BSA with either affinity-purified anti-Nup50 (∼0.5 μg/ml) or a mixture of anti-Nup50 and the mouse monoclonal antibody RL1 (∼1 μg/ml; Affinity BioReagents, Golden, Colo.), which recognizes several glycoproteins of the NPC ( ).

    Techniques: Isolation, Labeling