Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Intracellular localization and functional analysis of US11 protein. (A) Intracellular localization of US11 and nucleolin (NCL) in HeLa cells infected for 8 h with HSV-1 (wt 17+ strain, 10 PFU/cell) was visualized by indirect immunofluorescence using anti-US11 polyclonal and anti-nucleolin monoclonal antibodies. A merged image is presented. (B) Subcellular localization of wt US11 and Flag-US11 proteins in HeLa cells transfected with vectors expressing either untagged US11 (upper panels) or Flag-US11 fusion protein (lower panels). At 48 h after transfection, cells were fixed and the localization of the proteins was assayed with a polyclonal anti-US11 antibody and a monoclonal anti-nucleolin antibody (upper panels) or with a monoclonal anti-Flag antibody and a polyclonal anti-US11 antibody (lower panels). Merged images are presented. Scale bars represent 5 μm. (C) Transactivation of HTLV-I envelope glycoprotein expression by Rex, wt US11, and Flag-US11 proteins. HeLa cells were transfected with vectors expressing HTLV-env and Tax plus vectors expressing HTLV-Rex (upper left panel) or US11 (upper middle panel) or Flag-US11 fusion protein (upper right) or Flag only (lower left panel). Transactivation of the expression of HTLV-I env by either Rex or wt US11 led to the formation of syncytia (arrows in left and middle upper panels) as described in detail previously (10). The same transactivating property was displayed by Flag-US11, as visualized by syncytium formation (upper right panel). As control experiments, HeLa cells were either left untransfected (lower right panel) or transfected with vectors expressing HTLV-env and Tax plus vectors expressing either Flag only (lower left panel) or no additional vector (lower middle panel). As expected, in the three lower panels, only individual cells are visible and no syncytium was detected.

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Functional Assay, Infection, Immunofluorescence, Bioprocessing, Transfection, Expressing, Control, Plasmid Preparation

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Nucleolin depletion leads to US11 accumulation in nucleoli. (A and B) Immunofluorescence in a HeLa cell line stably expressing untagged US11 (clone HL5a1) transfected with scramble siRNA (SC) (A) or siRNA against nucleolin (B). Nucleolin (NCL) is shown in red and US11 in green, and DNA is stained with DAPI (blue). Scale bars represent 5 μm. (C and D) Fluorescence intensity profiles (gray level values) obtained on 16-bit images along the white lines indicated in panels A and B, respectively. Profiles correspond to nucleolin (red), US11 (green), and DAPI (blue) fluorescence intensities. (E) Quantification of the nucleolar/cytoplasmic ratio of US11. The ratio of nucleolar to cytoplasmic US11 fluorescence intensity was calculated for control untransfected cells (C; n = 18), mock-transfected cells (MT; n = 24), cells transfected with scramble siRNA (SC; n = 16), and cells transfected with nucleolin siRNA (NCL; n = 29). Five to 10 fields of view were analyzed for each set of conditions. Error bars correspond to standard deviations. Mann-Whitney and Wilcoxon statistical tests showed that the US11 ratio in nucleolin-depleted cells was significantly higher than in all the control cell populations (P < 2 × 10−7). (F) US11 cellular intensity in control untransfected cells (C), mock-transfected cells (MT), cells transfected with scramble siRNA (SC), and cells transfected with nucleolin siRNA (NCL) (camera gray level).

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Transfection, Staining, Fluorescence, Control, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Nucleolin is associated with Flag-US11 fusion protein in transfected cells: purification of the Flag-US11-containing complexes. (A) Optimization of the coimmunoprecipitation conditions of the Flag-US11 binding partners. HeLa cells were transfected with pSG5-Flag (lanes 1, 3, 5, 7, and 9) or with pSG5-Flag-US11 (lanes 2, 4, 6, 8, and 10) expression vectors. (Upper panel) Effect of NaCl concentration on the isolation of protein complexes containing Flag-US11 protein in a coimmunoprecipitation experiment using an anti-Flag antibody. Protein complexes were purified with an optimized buffer (see Materials and Methods) in the presence of an anti-Flag antibody and of various NaCl concentrations as indicated on the figure, and they were then analyzed by 1-DE and silver staining. The positions of the Flag-US11 protein and of molecular mass markers are indicated on the left. (Lower panel) The presence of nucleolin (NCL) in the corresponding purified complexes was checked by Western blot analysis using a polyclonal anti-nucleolin antibody. (B) Identification of nucleolin as a Flag-US11-interacting protein. Protein complexes (purified in the presence of 150 mM NaCl as described for panel A) from cells transfected with a plasmid expressing either Flag or Flag-US11 were separated by 1-DE and stained with Coomassie blue (upper panel). The portion of the gel corresponding to proteins of about 100 kDa in size (arrow on the right) was cut out, and the corresponding nucleolin protein was identified by mass spectrometry and bioinformatics analyses (see Table 1). The identity of this protein in the purified complexes was confirmed by Western blot analysis using a specific anti-nucleolin antibody (lower panel). The position of nucleolin (NCL) is indicated by an arrow on the right.

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Transfection, Purification, Binding Assay, Expressing, Concentration Assay, Isolation, Silver Staining, Western Blot, Plasmid Preparation, Staining, Mass Spectrometry

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Nucleolin is present in Flag-US11-containing complexes purified from transfected cells a

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Purification, Transfection, Sequencing

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Nucleolin interacts in vitro with full-length US11. (A) Nucleolin purified from CHO cells was biotinylated as previously described (15) and used for interaction studies with E. coli-expressed GST (lanes 1 to 3) or GST-US11 (lanes 4 to 6) proteins. Streptavidin beads (Strept. beads) were incubated with GST (lanes 2 and 3) or GST-US11 (lanes 5 and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of biotinylated nucleolin. After extensive washing, streptavidin beads were incubated in Laemmli loading buffer, and recovered proteins were separated by 1-DE and then visualized by silver staining. The positions of nucleolin and GST-US11 are indicated. (B) Nucleolin-p50 protein was produced in E. coli and purified as previously described (36). Glutathione Sepharose beads were incubated with binding buffer (lane 1), GST-US11 (lanes 2 and 3), or GST protein (lanes 6 and 7). Nucleolin-p50 protein was then added to the beads (lanes 1, 3, and 7). After extensive washing, beads were incubated with Laemmli buffer and proteins were separated by 1-DE and then visualized by silver staining.

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: In Vitro, Purification, Incubation, Silver Staining, Produced, Binding Assay

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: Interaction of nucleolin with US11 mutants. (A) Schematic representation of the different GST-US11 mutants used in this study. (B) The different proteins (1 μg) were separated by 1-DE, transferred on a nitrocellulose membrane, and then stained with Ponceau dye. The nucleolin-p50 protein (lane 8) corresponding to the C-terminal domain of nucleolin was used as a positive control for the Western blot analysis with anti-nucleolin antibody. (C) The membrane was incubated with the purified nucleolin protein as described in Materials and Methods, and after extensive washing, it was subjected to Western blot analysis with a polyclonal anti-nucleolin antibody. Nucleolin detection was then revealed using enhanced chemiluminescence (ECL). (D) As a control, a membrane similar to the one shown in panel B was subjected directly to a Western blot analysis with the anti-nucleolin antibody. Only the p50 nucleolin protein could be detected, showing that the nucleolin antibody does not cross-react with any of the GST-US11 proteins.

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Membrane, Staining, Positive Control, Western Blot, Incubation, Purification, Control

Journal: Journal of Virology

Article Title: Nucleolin Interacts with US11 Protein of Herpes Simplex Virus 1 and Is Involved in Its Trafficking

doi: 10.1128/JVI.06194-11

Figure Lengend Snippet: US11 is present in nucleolin-containing complexes purified from extracts of HSV-1-infected (HSV-1 inf.) cells. HeLa cells were mock infected at 0 h (lanes 1, 4, 7, and 10) or infected with HSV-1 for 8 h (lanes 2, 5, 8, and 11) and 16 h (lanes 3, 6, 9, and 12) at 10 PFU/cell. Coimmunoprecipitations (Co-IP) (lanes 1 to 9) were performed with the corresponding total cell extract proteins and used a polyclonal anti-nucleolin (anti-NCL; lanes 7 to 9) or an anti-HA (lanes 4 to 6) antibody or a preimmune serum (lanes 1 to 3), as indicated at the top of the figure. The presence of nucleolin (A) and of US11 (B) in the purified complexes was checked by Western blot analysis using anti-nucleolin- and anti-US11-specific antibodies, respectively. The positions of nucleolin and of US11 are indicated. Total cell extract proteins not submitted to the coimmunoprecipitation were used as positive controls (lanes 10 to 12).

Article Snippet: Rabbit polyclonal antibodies for US11 ( 12 ) or nucleolin ( 15 ) and mouse monoclonal anti-nucleolin (4E2 Assay Designs; Immunogen) (human nucleolin; 1/1,000 dilution) or M2 anti-Flag antibodies were the primary antibodies used.

Techniques: Purification, Infection, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

doi: 10.1074/jbc.m513406200

Figure Lengend Snippet: FIGURE 2. Nucleolin interacts with the tripartite palindrome motif of the KLF2 promoter in macrophages and endothelial cells. A, EMSAs were performed with nuclear extract from the WR19M.1 mouse macrophage cell line incubated with [-32P]ATP-labeled double-stranded oligo- nucleotide spanning the tripartite 30-bp palin- drome (157/95) region of the KLF2 promoter. Lane 1, probe alone; lane 2, input, no antibody; lane 3, 1000 competition with unlabeled probe; lane 4, anti-nucleolin polyclonal antibody; lane 5, nonspecific IgG antibody. B, EMSAs performed as inAwithnuclearextractfromstatic(left)andshear stressed (right) EOMA cells. The shear stress-spe- cific band (*) immunodepleted by the addition of anti-nucleolin antibody (arrow) is marked. C, immunoblot of EOMA whole cell lysate (30 g) under static conditions and after 6 and 12 h of fluid shear stress (19 dynes/cm2). The blot was first probed with anti-nucleolin polyclonal antibody and then stripped and reprobed with monoclonal anti-GAPDH. D, EMSA performed as in B using nuclear extract from static and sheared HUVECs. For immunodepletions (lanes 4 and 8), a mouse monoclonal anti-nucleolin antibody (clone 4i51) was used.

Article Snippet: Immunoprecipitations were performed in cell lysis buffer with 250–500 g of cell extract incubated for 4 h with rabbit anti-hnRNP-D (AUF1) antibody (Upstate Biotechnology, Inc.), mouse anti-nucleolin monoclonal antibody (Clone 4E2; Stressgen Bioreagents, Victoria, Canada), rabbit anti-hemagglutinin probe (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit anti-p85 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Incubation, Labeling, Shear, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

doi: 10.1074/jbc.m513406200

Figure Lengend Snippet: FIGURE 3. Binding of nucleolin to the KLF2 promoter in endothelial cells is dependent upon fluid flow and PI3K. A, EMSA performed with oligonucleotide probe of the 30-bp tripartite palindrome motif of the KLF2 promoter with nuclear extract isolated from EOMA cells exposed to 12 h of fluid shear stress (19 dynes/cm2) in the presence of LY294002 (40 mol/liter) or Me2SO (DMSO; vehicle) control. Complexes were competed with 1000 cold oligonucleotide for specificity. B, ChIP assays were performed on EOMA cells under static conditions(flow)andundershearstressconditions(flow;equalto19dynes/cm2for12h)inthepresenceofthePI3KinhibitorLY294002(40mol/liter)orMe2SO(vehiclecontrol). Immunoprecipitation was performed with anti-nucleolin polyclonal antibody. DNA amplification was performed with primers specific for the shear stress response region of the KLF2 promoter. Lanes 1–4, negative controls. Lane 1, no DNA PCR; lane 2, no chromatin mock ChIP; lane 3, no antibody immunoprecipitation; lane 4, nonspecific antibody immunopre- cipitation. Samples in lanes 5–9 are as marked. N.T., no treatment. C, ChIP assays were repeated on HUVECs with anti-nucleolin antibody under static (flow ) and shear stress conditions (flow ; equal to 19 dynes/cm2 for 12 h) in the presence of LY294002 (40 mol/liter) or Me2SO (vehicle) control. Lanes 1–4, negative controls (as above). N.T., no treatment.

Article Snippet: Immunoprecipitations were performed in cell lysis buffer with 250–500 g of cell extract incubated for 4 h with rabbit anti-hnRNP-D (AUF1) antibody (Upstate Biotechnology, Inc.), mouse anti-nucleolin monoclonal antibody (Clone 4E2; Stressgen Bioreagents, Victoria, Canada), rabbit anti-hemagglutinin probe (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit anti-p85 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Binding Assay, Isolation, Shear, Control, Immunoprecipitation, DNA Amplification

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

doi: 10.1074/jbc.m513406200

Figure Lengend Snippet: FIGURE4.Fluidflowresultsincatalysisofnucleo- lin. A, immunoblot with anti-nucleolin polyclonal antibody of 30 g of whole cell lysate from HUVEC cellskeptunderstaticconditions,orexposedto12h of fluid shear stress (19 dynes/cm2), in the presence or absence of LY294002 (40 mol/liter). First lane, static; second lane, flow; third lane, flow plus LY294002; fourth lane, flow plus Me2SO (DMSO). Samples were run on 10% polyacrylamide gel. The blot was stripped and reprobed with anti-GAPDH as aloadingcontrol.B,immunoblotwithmousemono- clonal anti-nucleolin antibody (clone 4E2), using 20 g of static or shear-stressed (12 h) HUVEC whole cell lysate run on a 4–15% polyacrylamide gel. C, immunoblotwithmousemonoclonalanti-nucleolin antibody (clone 4i51) using 10 g of static or shear- stressed (6 h) HUVEC whole cell lysate run on a 10% polyacrylamide gel. D, immunoblot shear stress (19 dyne/cm2) time course of EOMA whole cell extract run on 10% polyacrylamide gel and probed with anti-nucleolin polyclonal antibody. Lane 1, static; lane2,5min;lane3,1h;lane4,12h;lane5,24h;lane 6, 12 h plus LY294002 (40 mol/liter); lane 7, 12 h plusMe2SO.TheblotwasreprobedwithGAPDHasa loading control.

Article Snippet: Immunoprecipitations were performed in cell lysis buffer with 250–500 g of cell extract incubated for 4 h with rabbit anti-hnRNP-D (AUF1) antibody (Upstate Biotechnology, Inc.), mouse anti-nucleolin monoclonal antibody (Clone 4E2; Stressgen Bioreagents, Victoria, Canada), rabbit anti-hemagglutinin probe (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit anti-p85 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Western Blot, Shear, Control

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

doi: 10.1074/jbc.m513406200

Figure Lengend Snippet: FIGURE 5. Nucleolin interacts with the p85 regulatory subunit of PI3K and hnRNP-D. A, immunoprecipitation (IP) of HUVECs transiently transfected with HA-tagged nucleolin expression vector. 24 h after transfection, the cells were exposed to 12 h of fluid shear stress in the presence of LY294002 (40 mol/liter) or Me2SO (vehicle) control. Protein complexes were precipitated with anti-HA antibody, followed by 10% polyacrylamide gel electrophoresis and immunoblotting (IB) with anti-p85 antibody. Blots were stripped and reprobed with anti-HA antibody as an internal control. B, IP of static or shear-stressed (12 h at 19 dynes/cm2) HUVECs with anti-p85 antibody, followed by immunoblotting with anti-nucleolin monoclonal antibody. Blots were stripped and reprobed with anti-p85 antibody for internal control. C, IP of static or shear stressed HUVECs, in the presence of LY294002 (40 mol/liter) or Me2SO, with anti-nucleolin monoclonal (clone 4E2) antibody followed by immunoblotting with anti-hnRNP-D antibody. Blots were stripped and reprobed with anti-nucleolin antibody. D, IP of HUVECs (under static, shear-stressed, or shear-stressed plus LY294002 conditions) with anti-hnRNP-D antibody, followed by immunoblotting with anti-nucleolin antibody. Blots were stripped and reprobed with anti-hnRNP-D antibody.

Article Snippet: Immunoprecipitations were performed in cell lysis buffer with 250–500 g of cell extract incubated for 4 h with rabbit anti-hnRNP-D (AUF1) antibody (Upstate Biotechnology, Inc.), mouse anti-nucleolin monoclonal antibody (Clone 4E2; Stressgen Bioreagents, Victoria, Canada), rabbit anti-hemagglutinin probe (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit anti-p85 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Shear, Control, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

doi: 10.1074/jbc.m513406200

Figure Lengend Snippet: FIGURE 6. Inhibition of nucleolin by siRNA blocks the induction of KLF2 mRNA by fluid shear stress. A, HUVECs were transfected with functional nontargeting siRNAs (F.N.T., negative control), lamin A/C siRNA (positive control), or nucleolin siRNA. Whole cell lysate was isolated 48 h after transfection, and 30 g was run on 10% polyacrylamide gel. Blots were probed with anti-nucleolin polyclonal antibody, stripped, and reprobed with anti-lamin A/C antibody and then stripped and reprobed with anti-GAPDH antibody. B, the effect of nucleolin reduction on flow-mediated induction of KLF2 was determined by reverse transcription-PCR. HUVECs were transfected with functional nontargeting siRNA or nucleolin siRNA and allowed to recover overnight. Cells were then exposed to 12 h of fluid shear stress (19 dynes/cm2). RNA was isolated and reversed transcribed, and PCR was performed (30 cycles) with gene-specific primers for KLF2, nucleolin, or GAPDH.

Article Snippet: Immunoprecipitations were performed in cell lysis buffer with 250–500 g of cell extract incubated for 4 h with rabbit anti-hnRNP-D (AUF1) antibody (Upstate Biotechnology, Inc.), mouse anti-nucleolin monoclonal antibody (Clone 4E2; Stressgen Bioreagents, Victoria, Canada), rabbit anti-hemagglutinin probe (Y-11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit anti-p85 polyclonal antibody (Cell Signaling Technology, Inc., Beverly, MA).

Techniques: Inhibition, Shear, Transfection, Functional Assay, Negative Control, Positive Control, Isolation, Reverse Transcription