Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 2. Inhibition of nuclear capsid binding in syringe-loaded Vero cells. (A) Fluorescence micrographs of syringe-loaded Vero cells in- fected with HSV-1 K26GFP. Note that accumulations of capsids are present at the nuclear surface of cells loaded with the following anti- bodies: control, VP23, VP5, UL37, and Nup214 (arrows). (B) Graph showing the number of nucleus-bound capsids as a percentage of control in cells syringe-loaded with herpesvirus antibodies (anti-VP5, anti-VP23, anti-VP1/2, and anti-UL37). (C) Graph of nuclear capsid binding inhibition in cells loaded with nucleoporin antibodies (anti- Nup358 and anti-Nup214). In panels B and C, bars represent means of duplicate or triplicate (VP1/2 and Nup358) experiments with the stan- dard error shown (*, P 0.0041; **, P 0.0001). An average of between 40 and 50 cells were analyzed per sample per experiment. Cells were infected 3 to 4 h after antibody loading. Nuclear binding was assessed 3 h postinfection.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Inhibition, Binding Assay, Fluorescence, Control, Infection

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 3. Immunofluorescent labeling of herpesvirus proteins and nucleo- porins. (A and B) Fluorescence micrographs of cells infected with K26GFP and immunolabeled for VP5, VP23, VP1/2, and UL37. Vero cells were infected with HSV-1 for 3 h, immunolabeled with antibodies against HSV-1 proteins, and visualized with Alexa 594-conjugated secondary antibodies. Capsids were detected by GFP signal. Note that GFP and protein labels occur coincidently at the cell surface (arrowheads) and at the nuclear surface (ar- rows) for VP5, VP23, VP1/2, and UL37. Scale bar, 10 m. Cells in panel A were fixed prior to labeling, while cells in panel B were fixed after labeling. DAPI, 4,6-diamidino-2-phenylindole. (C) Unfixed cells were immunola- beled with nucleoporin antibodies. Both Nup358 and Nup214 antibodies exhibited a nuclear staining pattern consistent with interaction with their target antigen. Scale bar, 10 m.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Labeling, Fluorescence, Infection, Immunolabeling, Staining

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 4. Nucleoporin removal by siRNA treatment. (A) Fluores- cence micrographs of immunolabeled Vero cells transfected with siRNAs specific for one of three nucleoporins: cytoplasmic filament protein Nup358, cytoplasmic facing protein Nup214, or nuclear basket protein TPR. Panels show nucleoporin signal (red) in siRNA-treated cells relative to signal in untreated cells. Treatment is indicated in the bottom left corner of each image, and staining is indicated at the top of each image column. (B to D) Western blots with importin- serving as a loading control. Samples were taken 72 and 96 h posttransfection. “s” indicates cells transfected once. “d” indicates cells transfected twice, at 0 and 24 h post-initial transfection. (B) Western blot of Vero cells transfected with Nup358 siRNA alone and Nup358 siRNA plus Nup214 siRNA. (C) Western blot of Vero cells transfected with TPR siRNA. (D) Western blot of Vero cells transfected with Nup214 siRNA.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Immunolabeling, Transfection, Staining, Western Blot, Control

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 5. Nup214 and Nup358 localization in siRNA-treated Vero cells. Fluorescence micrographs show nuclear localization patterns of Nup214 and Nup358 with three different treatments. Vero cells were transfected with siRNA specific for Nup214 or Nup358 or were given no treatment. Seventy-two hours after transfection, cells were fixed and immunostained for Nup214 and Nup358. Treatment is indicated to the left of the image row. Label is indicated at the top of the column of images. Note that untreated cells exhibit nuclear staining for both Nup358 and Nup214. Nuclear Nup358 and Nup214 staining are greatly reduced in Nup214 siRNA-treated cells. Nuclear Nup358 but not Nup214 is reduced in cells treated with Nup358 siRNA. DAPI, 4,6- diamidino-2-phenylindole. Scale bar, 10 m.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Fluorescence, Transfection, Staining

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 6. Nuclear capsid binding in siRNA-treated cells. (A) Fluorescence micrographs of immunolabeled and 4,6-diamidino-2-phenylindole- stained siRNA-treated Vero cells infected with HSV-1 K26GFP. The siRNA treatment is indicated in the top left corner of each image. The cellular protein stained red is indicated in the bottom left. Capsids appear green. White arrows indicate accumulations of capsids at the nuclear surface 3 h postinfection. Scale bar, 10 m. (B) Graph of Nup358 protein concentration (dark gray) and nuclear capsid binding (light gray) with standard error shown (*, P 0.0025; **, P 0.0001; **, P 0.002). Note that nuclear capsid binding reduction corresponds to Nup358 protein concentration reduction except in the case of Nup214 siRNA, in which capsid binding is reduced in the absence of Nup358 protein reduction.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Binding Assay, Fluorescence, Immunolabeling, Staining, Infection, Protein Concentration

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 7. Colocalization of herpesvirus capsids with Nup358 and Nup214. (A) Immunofluorescence of HSV-1 K26GFP-infected Vero cells. Cells were infected for 3 h and then fixed and stained for nucleo- porins (red Nup214 in panels a and c or red Nup358 in panels b and d). High magnification of nuclear capsids is shown in panels c and d. A greater degree of colocalization was seen between capsids and Nup358 (panels b and d) than between capsid and Nup214 (panels a and c). Scale bar, 5 m (panels a to d). (B) Graph of percent colocalization between capsids and nucleoporins. Each bar represents the number of green nuclear pixels overlapping with red nuclear pixels divided by the total number of green nuclear pixels, expressed as a percentage.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

doi: 10.1128/jvi.01139-08

Figure Lengend Snippet: FIG. 8. Model of capsid binding at the nucleus. The model depicts the fate of HSV-1 capsids in newly infected cells. After fusion at the plasma membrane, the capsid and tegument enter the cytoplasm. Upon entry, loosely associated tegument is separated from the capsid, while a subset of tightly associated tegument proteins (VP1/2, UL37) remain attached as the capsid transits to the nucleus. Once at the nucleus, a vertex resident protein, possibly VP1/2, interacts with the pore to anchor the capsid with its distinctive vertex-to-pore orientation. Two nuclear pore proteins are highlighted in this model: Nup358 and Nup214. Herpesvirus capsids are shown bound to Nup358, the putative cytoplasmic filament protein.

Article Snippet: For immunofluorescence, antibodies were used at the following dilutions: VP5, 1:250; VP23, 1:500; VP1/2, 1:250; UL37, 1:300; Nup358, 1:250; Nup214, 1:250; and TPR (1A8, Novus Biologicals), 1:600.

Techniques: Binding Assay, Infection, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: A. Constant total protein from siRNA-treated cultures was analyzed for nucleoporin content by immunoblot with the indicated antibodies. Dilutions of the cultures treated with control siRNA are shown for comparison to semi-quantify the extent of knockdown (siRNA knockdown resulted in approximately the same signal intensity as dilution to 25%). B. Immunofluorescence microscopy of TOV112D cells treated with NUP62 or NUP214 siRNAs. Immunofluorescent images represent 3D deconvolved projections of 10–15 um total of optical sections through the z axis. C. Immunofluorescence microscopy of NUP133 and NUP62 or NUP214 in TOV112D cells. Bars represent 2 um.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Western Blot, Control, Comparison, Knockdown, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: Whole cell, cytosolic, and nuclear fractions from Hs 832(C).T (Hs), TOV112D, HEK293 (293), S12, and COS7 cells were analyzed by immunoblot with antibodies to NUP62 and NUP214 (antibodies used were also used for immunofluorescence). Two T75 plates of cells at 80% confluence were used for each preparation (this does not yield equal numbers of cells). One third of the sample was taken for the whole cell extract; the remaining sample was fractionated into cytosol and nuclear components. Extracts were loaded to represent equivalent fractions of starting material. Indicated as Mr∼35K is an additional band observed in the NUP214 immunoblot. The Mr∼70K band present in the Hs832(C).(Hs) whole cell lysate fractionated with the cytosol. The Western blot analyses of the whole cell lysates were completed on single membranes for all five cell lines; however, the exposure times for Hs832(C).(Hs) and Cos 7 cells using the NUP214 antibody and for Hs832(C).(Hs) using the NUP62 antibody were longer. Consequently, those lanes are separated.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Western Blot, Immunofluorescence

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: HEK293 and TOV112D cells were immunolabeled with NUP62 (green) and NUP214 (red) antibodies. Optical sections were generated from the culture plate surface upward to the medium at intervals of 250 nm. The first focused nuclear surface plane (0 um), a central plane, and last focused surface plane were selected and 2D deconvolved. Overlay of NUP62 and NUP214 signals is shown, with coincident signals revealed in yellow. Blue, DAPI stain of nuclei. Bar represents 5 um.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Immunolabeling, Generated, Staining

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 10 um. A. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP62 and cultured for 72 hours. The distribution of NUP62 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP62. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP62 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distribution of NUP214 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. The images of the pair of post-mitotic cells shown have been cropped individually to avoid interference from surrounding cells, particularly on the x,z and z,y planes. Immunofluorescence analyses of NUP214 (red) and V5 epitope-tagged NUP214 (green) were performed with Ab4 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. C. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distributions of NUP62 and NUP214 were analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP214 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Transfection, Expressing, Plasmid Preparation, Cell Culture, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: A. TOV112D cells were treated in the absence of exogenous DNA with Lipo293D (SignaGen Laboratories), Lipofectamine (Invitrogen), or FuGENE HD (Promega), according to the instructions provided by the manufacturers. The cells were processed for immunofluorescence using NUP62 (green) and NUP214 (red) antibodies 48 hours after treatment. Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 5 um. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. C. TOV21G cells were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. D. TOV21G cell cultured at low density and analyzed by immunofluorescence microscopy as described above.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Immunofluorescence, Staining, Cell Culture, Microscopy

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: Top left corner: immunoblot analyses of Hs 832(C).T (Hs), TOV112D (112D), HEK293 (293), S12, and COS7 (cos7) cell lysates. Antibodies are explained in the text. Immunofluorescent images for the indicated cell lines represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. immunofluorescence analyses of NUP62 (green) and NUP214 (red) were generated using Ab1 and Ab4, respectively. Blue, DAPI stain; yellow, overlap between green and red. Bars represent 2 um.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Western Blot, Immunofluorescence, Generated, Staining

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: Double immunofluorescence analyses of NUP62 (green) and NUP214 (red) are shown for large fields of subconfluent TOV112D ovarian carcinoma, COS7, S12 neuroblastoma, and HEK293 cells. Blue, DAPI stain; yellow, overlap between green and red. Bar represent 10 um.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: Immunofluorescent images for fields of TOV112D, HEK293 (293), COS7, or S12 cells (similar to those shown in ), and images from several different Hs 832(C).T cells, were analyzed. Radial intensity distributions for NUP62 or NUP214 were determined for each cell line and normalized to arc length, and a minimum of 24 sectors derived from at least six independent nuclei were used for each analysis. Radial distances were normalized from 0 (point of origin) to 1 (surface of nuclear envelope). Distribution points are colored according to residuals (blue: less than one standard error; green: less than two standard errors; red: greater than two standard errors). Plotted in red are 95% confidence intervals.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: HIgh resolution STED (NUP214; red) and standard confocal (NUP62; green) double immunofluorescence microscopy were performed on S12 neuroblastoma cells. Individual optical sections were selected from the ventral (culture plate side) nuclear surface (0 um) upward to the dorsal surface at intervals of 250 nm. Yellow, overlap bewteen green and red. Bar represents 5 um.

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Nuclear Distributions of NUP62 and NUP214 Suggest Architectural Diversity and Spatial Patterning among Nuclear Pore Complexes

doi: 10.1371/journal.pone.0036137

Figure Lengend Snippet: HIgh resolution STED microscopy (STED) and confocal (Conf.) double immunofluorescence microscopy were performed with NUP62 (green) and NUP214 (red) antibodies in S12 neuroblastoma cells. All images are projections of 8–10 um total optical z-stacks. Bars represent 500 nm. A. Comparison of confocal and STED microscopy resolution of NUP214 antibody immunofluorescence. A and B. Arrows, NUP62 + /NUP214 − NPCs; asterisks, NUP62>NUP214 NPCs; diamonds, NUP62 + /NUP214 + NPCs; triangles, NUP62

Article Snippet: Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC-00103 (Ab4) and A300–717A (Ab3)).

Techniques: Microscopy, Immunofluorescence, Comparison, Derivative Assay

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Oxidative stress changes the levels of Crm1 and multiple nucleoporins at the nuclear envelope. (A) The association of Crm1, Nup358, Nup214, Nup62, and RanGAP1 with nuclear envelopes was quantified under control and stress conditions by using the multiwavelength translocation module. A drastic reduction in nuclear envelope fluorescence is observed for Nup358. (B) Stress-induced changes in Crm1 associated with the cytoplasmic side of the NE. Control and DEM-treated cells were fixed and semipermeabilized with digitonin (Kodiha et al., 2008b ) to detect Crm1 at the cytoplasmic face of the NE. Bar, 20 μm.

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: Control, Translocation Assay, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Analysis of stress-induced shifts in Nup214 and Nup62 electrophoretic mobility. (A) Nup62, Nup214, and Nup98 are phosphorylated under normal and stress conditions. Crude extracts prepared from control or DEM-treated cells were incubated with CIP and examined by Western blotting. (B) Oxidative stress increases the O-GlcNAc modification of Nup62 and Nup214. Crude extracts from untreated or stressed cells were digested with hexosaminidase (Hex) and analyzed by Western blotting. (C) Stress-induced changes in O-GlcNAc modification were quantified for Nup62 and Nup214. Nucleoporins were immunoprecipitated under denaturing conditions, followed by Western blotting with antibodies against O-GlcNAc moieties. Filters were stripped and reprobed to measure the amount of nucleoporin. ECL signals were determined by densitometry following standard procedures. For control samples, the ratio GlcNAc/nucleoporin was defined as 1. Note that the O-glycosylation of both Nup62 and Nup214 increased in response to DEM treatment. Student's t test, *p < 0.05.

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: Control, Incubation, Western Blot, Modification, Immunoprecipitation, Glycoproteomics

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Oxidative stress increases the association between Crm1 and Nup62, Nup153 or Ran, but reduces Nup88/Crm1 and Nup88/Nup214 complexes. Proteins in control (EtOH) and stressed cells (DEM) were covalently cross-linked with DSP. Crude extracts were prepared for immunoprecipitation (IP) with antibodies against the proteins indicated, and immunopurified proteins were analyzed by Western blotting as described in Materials and Methods. (A) ECL signals were quantified to determine the ratio Nup62/Crm1 for immunoprecipitations with Crm1, and the ratio Crm1/Nup62 for immunoprecipitation with Nup62 specific antibodies. Results are shown for three independent experiments, the ratio of Nup62/Crm1 or Crm1/Nup62 was defined as 1 for control samples. (B and C) Coimmunoprecipitations with antibodies against Crm1, Nup153, or Nup214 were carried out and analyzed as described in A. (D) Immunocomplexes were isolated with antibodies against Crm1 or Nup214 and probed for the presence of Nup88. (E) The interaction between Crm1 and Ran was tested under normal and stress conditions. Significant differences in binding under normal and stress conditions were identified with Student's t test, *p < 0.05 and **p < 0.01. Note that the associations Nup62/Crm1, Nup153/Crm1, and Ran/Crm1 increased in DEM-treated cells, whereas a reduction was observed for Nup88/Crm1 and Nup88/Nup214.

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: Control, Immunoprecipitation, Western Blot, Isolation, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Oxidant treatment alters the interactions of Nup62 with Nup214 and Nup153. DSP–cross-linked proteins from control and DEM-treated samples were immunoprecipitated with antibodies against different nucleoporins as shown in the figure. ECL signals of Western blots were evaluated as described for Figure 6. In these experiments, interactions between Nup62 and Nup214 or Nup153 were reduced by stress.

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: Control, Immunoprecipitation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Oxidative stress affects multiple components of the nuclear export apparatus

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: Modification, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

doi: 10.1091/mbc.E09-05-0397

Figure Lengend Snippet: Simplified model for oxidant-induced changes at the NPC. Oxidative stress affects Crm1 (brown), Nup62 (red), and Nup214 (gray) at the NE. Oxidant exposure enhances the interaction between Crm1 and Nup62, while the posttranslational modifications of Nup62 and Nup214 increase. See Discussion for details.

Article Snippet: Denatured proteins were immunoprecipitated with antibodies specific for Nup62 or Nup214 (catalog nos. sc-1915 and sc-26055, Santa Cruz Biotechnology; catalog no. 1-97, Abnova), Ran (catalog no. sc-1156, Santa Cruz Biotechnology), and mab414 (BAbCO, Richmond, CA) essentially as described previously ( Kodiha et al. , 2004 ).

Techniques: