primary anti nucleolin polyclonal antibody  (Thermo Fisher)

Journal: PLoS ONE

Article Title: The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals

doi: 10.1371/journal.pone.0221505

Figure Lengend Snippet: (A) Microscopy analysis of EGFP-JDV Rev WT protein (in green) expressed in MDBK and BoMac cells following transfection for 24 h with pEGFP-JDV Rev WT. Cells were left untreated or treated with 5 nM of leptomycin B (LMB) for 5 h and then fixed, subjected to immunostaining for nucleolin detection (in red) and counterstained with DAPI for nucleus visualization (in blue). The merge panel represents the superposition of EGFP-JDV Rev, DAPI, and nucleolin images. The images shown are representative of the expression pattern observed in cells from three independent experiments. The white bars correspond to a length of 10 μm. Images were derived by using CLSM at 60x magnification and analyzed to determine the Fn/c (B) and the Fno/n (C) ratios. Results are expressed as the mean Fn/c or Fno/n ratio ± SEM ( n = 30). For each cell line, significant differences, using a Student’s T-test corrected with the Holm-Sidak method for multiple comparison of the means, with and without LMB treatment, are indicated by ** ( P < 0.005) and *** ( P < 0.0005). No significant differences, using an ANOVA Tuckey’s multiple-comparison test, were observed between the Fn/c and Fno/n ratios measured for the MDBK and BoMac cells, regardless of the LMB treatment.

Article Snippet: After an incubation of 24 h and, where indicated, 5 nM of leptomycin B (LMB), a known nuclear exit inhibitor [ ], was added to the cell culture medium for 5 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) solution for 15 min. For the immunofluorescence assay, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% bovine serum albumin in PBS for 1 h at 37°C, and then incubated with rabbit polyclonal IgG primary anti-C23 (nucleolin) antibodies (H-250) (Santa Cruz Biotechnologies, Dallas, TX) for 1 h at 37°C.

Techniques: Microscopy, Transfection, Immunostaining, Expressing, Derivative Assay, Comparison

Journal: PLoS ONE

Article Title: The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals

doi: 10.1371/journal.pone.0221505

Figure Lengend Snippet: (A) JDV Rev mutant-encoding sequences were generated from the JDV Rev WT gene by PCR-ligation-PCR and then cloned into pEGFP-C1 for expression of the JDV Rev mutant (JM) proteins fused to EGFP. ARM: arginine-rich motif. (B) Microscopic analysis of the JM4 to JM6 mutant proteins (in green) expressed in MDBK cells at 24 h post transfection. Cells were fixed, subjected to immunostaining for nucleolin detection (in red) and counterstained with DAPI for nucleus visualization (in blue). Images were derived by using CLSM at 60x magnification and are representative of the expression pattern observed in cells from three independent experiments. The merge panel represents the superposition of EGFP-JDV Rev, DAPI, and nucleolin images. The while bars correspond to a length of 10 μm. CLSM images were analyzed to determine the Fn/c ratios ( C ) without or with LMB treatment. Results expressed as the mean Fn/c ratio ± SEM ( n = 30) are shown for the JDV Rev WT protein and each of the JDV Rev deletion mutants. Significant differences, using an ANOVA Dunnett’s test, between the JDV Rev WT protein and each of the deletion mutant proteins (JM1 to JM10) are indicated by ** ( P < 0.005). **** ( P < 0.00005). (D) The nuclear export activity of EGFP-JDV Rev WT or EGFP-Rev deletion mutant proteins (JM1 to JM10) was determined using a CAT reporter assay. The CAT expression levels were normalized to EGFP-Rev expression for each protein in cell lysates as determined by Western blot using an EGFP-specific antibody (bottom of the panel). Rev activity was then determined as the ratio of EGFP-JDV Rev WT or mutant protein CAT expression to the basal expression from pDM128 or pDM138 constructs co-transfected with empty pEGFP-C1 only. The mean Rev activity values + SEM were obtained from three independent experiments (triplicate samples per experiment). Significant differences, using an ANOVA Dunnett’s test, between the JDV Rev WT protein and each of the deletion mutant proteins are indicated by ** ( P < 0.005), and *** ( P < 0.0005). Unt: untransfected cells.

Article Snippet: After an incubation of 24 h and, where indicated, 5 nM of leptomycin B (LMB), a known nuclear exit inhibitor [ ], was added to the cell culture medium for 5 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) solution for 15 min. For the immunofluorescence assay, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% bovine serum albumin in PBS for 1 h at 37°C, and then incubated with rabbit polyclonal IgG primary anti-C23 (nucleolin) antibodies (H-250) (Santa Cruz Biotechnologies, Dallas, TX) for 1 h at 37°C.

Techniques: Mutagenesis, Generated, Ligation, Clone Assay, Expressing, Transfection, Immunostaining, Derivative Assay, Activity Assay, Reporter Assay, Western Blot, Construct

Journal: PLoS ONE

Article Title: The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals

doi: 10.1371/journal.pone.0221505

Figure Lengend Snippet: (A) The 74 to 105 aa sequence containing basic residues present in deleted sequences of JDV Rev mutants JM4 and JM5 was inserted into pEGFP-C1, pEGFP-βGal or pEGFP-GST vectors. (B) MDBK cells were transfected with plasmid vectors encoding either the EGFP, EGFP-βGal or EGFP-GST proteins alone or fused to the JDV 74-105 REV sequence described in panel A. After an incubation time of 24 h, the cells were fixed, subjected to immunostaining for nucleolin detection (in red) and counterstained with DAPI for nucleus visualization (in blue). Expression of the proteins was detected via the EGFP fluorescence (in green). Images were derived by using CLSM at 60x magnification and are representative of the expression pattern observed in cells from three independent experiments. The merge panel represents the superposition of EGFP, DAPI and nucleolin images. The white bars correspond to a length of 10 μm. (C) CLSM images were analyzed to determine the Fn/c ratios. Results (mean Fn/c ratio ± SEM, for n = 30) are shown for the different EGFP proteins. Significant differences, using a Student’s T-test, between EGFP, EGFP-GST or EGFP-βGal and each of the protein counterparts fused to the JDV 74-105 REV sequence, are indicated by ****( P < 0.00005).

Article Snippet: After an incubation of 24 h and, where indicated, 5 nM of leptomycin B (LMB), a known nuclear exit inhibitor [ ], was added to the cell culture medium for 5 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) solution for 15 min. For the immunofluorescence assay, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% bovine serum albumin in PBS for 1 h at 37°C, and then incubated with rabbit polyclonal IgG primary anti-C23 (nucleolin) antibodies (H-250) (Santa Cruz Biotechnologies, Dallas, TX) for 1 h at 37°C.

Techniques: Sequencing, Transfection, Plasmid Preparation, Incubation, Immunostaining, Expressing, Fluorescence, Derivative Assay

Journal: PLoS ONE

Article Title: The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals

doi: 10.1371/journal.pone.0221505

Figure Lengend Snippet: (A) JDV Rev arginine (R) to alanine (A) substitution mutant proteins (Mut1 to M17) were generated from pEGFP-JDV Rev WT. (B ) Subcellular localization of JDV Rev WT and mutant proteins harboring multiple arginine to alanine substitutions (Mut8 to Mut16) described in panel A. MDBK cells were transfected with pEGFP-JDV Rev WT or each of the mutant constructs, and incubated for 24 h. Cells were treated with leptomycin B (LMB) for 5 h or left untreated. Cells were then fixed, subjected to immunostaining for nucleolin detection (in red) and counterstained with DAPI for nucleus visualization (in blue). Images were derived by using CLSM at 60x magnification and are representative of the expression pattern observed in cells from three independent experiments. The merge panel represents the superposition of EGFP, DAPI and nucleolin images. The white bars correspond to a length of 10 μm. (C) CLSM images were analyzed to determine the Fn/c ratios. Results (mean Fn/c ratio ± SEM, for n = 30) are shown for the JDV Rev WT protein and each of the alanine substitution mutants. Significant differences, using an ANOVA Dunnett’s test, between the JDV Rev WT protein and each of the substitution mutant proteins with LMB treatment are indicated by ** ( P < 0.005), *** ( P < 0.0005), and **** ( P < 0.00005). (D) Nuclear export activity of the EGFP-JDV Rev WT and mutant proteins (Mut1 to Mut17) was determined using a CAT reporter assay. The CAT levels were normalized to the expression level of EGFP-JDV Rev WT or mutant proteins as determined by Western blot using an EGFP-specific antibody (bottom of the panel). Rev activity was then determined as the ratio of EGFP-JDV Rev WT or mutant protein CAT expression to the basal expression from pDM128 or pDM138 constructs co-transfected with empty pEGFP-C1 only. The mean Rev activity values + SEM were obtained from three independent experiments (triplicate samples per experiment). Significant differences, using an ANOVA Dunnett’s test, between the JDV Rev WT protein and each of the substitution mutant proteins are indicated by * ( P < 0.05) and ** ( P < 0.005). Unt: untransfected cells.

Article Snippet: After an incubation of 24 h and, where indicated, 5 nM of leptomycin B (LMB), a known nuclear exit inhibitor [ ], was added to the cell culture medium for 5 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) solution for 15 min. For the immunofluorescence assay, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% bovine serum albumin in PBS for 1 h at 37°C, and then incubated with rabbit polyclonal IgG primary anti-C23 (nucleolin) antibodies (H-250) (Santa Cruz Biotechnologies, Dallas, TX) for 1 h at 37°C.

Techniques: Mutagenesis, Generated, Transfection, Construct, Incubation, Immunostaining, Derivative Assay, Expressing, Activity Assay, Reporter Assay, Western Blot

Journal: PLoS ONE

Article Title: The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals

doi: 10.1371/journal.pone.0221505

Figure Lengend Snippet: (A) MDBK cells were transfected with plasmid constructs encoding either EGFP or EGFP fused, at its C-terminal, to each of the JDV Rev arginine cluster sequences shown in the figure. (B) After an incubation of 24 h the cells were fixed, subjected to immunostaining for nucleolin detection (in red) and counterstained with DAPI for nucleus visualization (in blue). Images were derived by using CLSM at 60x magnification and are representative of the expression pattern observed in cells from three independent experiments. The merge panel represents the superposition of EGFP, DAPI and nucleolin images. The white bars correspond to a length of 10 μm. (C) The CLSM images were analyzed to determine the Fn/c and Fno/n ratios. Results were expressed as the mean Fn/c or Fno/n ratio ± SEM ( n = 30). Significant differences between the EGFP proteins, using an ANOVA Tukey’s multiple-comparison test, are indicated by *** ( P < 0.0005) and **** ( P < 0.00005).

Article Snippet: After an incubation of 24 h and, where indicated, 5 nM of leptomycin B (LMB), a known nuclear exit inhibitor [ ], was added to the cell culture medium for 5 h. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.3) solution for 15 min. For the immunofluorescence assay, cells were permeabilized with 0.2% Triton X-100 for 10 min, blocked with 4% bovine serum albumin in PBS for 1 h at 37°C, and then incubated with rabbit polyclonal IgG primary anti-C23 (nucleolin) antibodies (H-250) (Santa Cruz Biotechnologies, Dallas, TX) for 1 h at 37°C.

Techniques: Transfection, Plasmid Preparation, Construct, Incubation, Immunostaining, Derivative Assay, Expressing, Comparison