Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal:

Article Title: Adjuvant Activities of Novel Cytokines, Interleukin-23 (IL-23) and IL-27, for Induction of Hepatitis C Virus-Specific Cytotoxic T Lymphocytes in HLA-A*0201 Transgenic Mice

doi: 10.1128/JVI.78.17.9093-9104.2004

Figure Lengend Snippet: Expression and activity of the scIL-12, scIL-23, and scIL-27 fusion proteins. (A) p3XFLAG-IL-12 (IL-12), p3XFLAG-IL-23 (IL-23), p3XFLAG-IL-27 (IL-27), and p3XFLAG (vector) were transiently transfected into 293T cells. Proteins secreted in the supernatants were immunoprecipitated with the anti-FLAG antibody. The immunoprecipitated proteins were separated on by SDS-12% polyacrylamide gel electrophoresis and subjected to Western blotting analysis with the anti-FLAG antibody. The positions of protein molecular mass markers (in kilodaltons) are shown in the figure, and the three arrows indicate the bands of the scIL-12, scIL-23, and scIL-27 fusion proteins. (B to D) STAT tyrosine phosphorylation assay. 293T cells were transiently transfected with either pME18S-IL-12Rβ1 plus pME18S-IL-12Rβ1 (B), pME18S-IL-12Rβ1 plus p3XFLAG-IL-23R (C), or p3XFLAG-WSX-1/TCCR plus p3XFLAG-gp130 (D). After 36 h, the cells were stimulated for 45 min with the culture supernatant containing either scIL-12 (IL-12) (B), scIL-23 (IL-23) (C), or scIL-27 (IL-27) (D) at final concentrations of 0, 0.5, 5, and 50%. As negative controls, the cells were stimulated for 45 min with the culture supernatant containing either scIL-23/scIL-27 (B), scIL-12/scIL-27 (C), or scIL-12/scIL-23 (D) at a final concentration of 50%. The cells were then subjected to Western blotting with anti-STAT1 (Total STAT1) (D), anti-STAT4 (Total STAT4) (B and C), anti-pY-STAT1 (pY-STAT1) (D), and anti-pY-STAT4 (pY-STAT4) (B and C) antibodies.

Article Snippet: The cells were then subjected to Western blotting with anti-STAT1, anti-STAT4 (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-pY-STAT1 (Cell Signaling Technology, Inc., Beverly, Mass.), or anti-pY-STAT4 (Zymed Laboratories, Inc., South San Francisco, Calif.) antibody.

Techniques: Expressing, Activity Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Phosphorylation Assay, Concentration Assay

Journal: medRxiv

Article Title: Neonatal-onset autoinflammation and immunodeficiency caused by heterozygous missense mutation of the proteasome subunit β-type 9

doi: 10.1101/2021.02.01.21250077

Figure Lengend Snippet: Panels A and B show the proteasome analysis of unstimulated or IFN-γ-stimulated skin fibroblasts from a healthy control and patient 2. Cell extracts were prepared and fractionated by glycerol gradient centrifugation. Panel A shows immunoblot analysis of each fraction using Abs against the indicated proteins. Panel B shows chymotrypsin-like activity of each fraction, which was measured by using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.0025% SDS. Panel C shows the STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts of control subjects and Patient 2. The expression levels of IFN-γ induced p-STAT1 were enhanced in patient 2 compared to control subjects. Panel D shows the skin biopsy sample of patient 2 stained with anti-ubiquitin Ab. A scale bar represents 100 μm.

Article Snippet: Six μm sections of biopsy samples from the patients’ skin lesion were stained with rabbit polyclonal anti-p-STAT1 Abs (GeneTex) and anti-ubiquitin Abs (DAKO).

Techniques: Control, Gradient Centrifugation, Western Blot, Activity Assay, Phospho-proteomics, Transformation Assay, Expressing, Staining, Ubiquitin Proteomics

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: bioRxiv

Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

doi: 10.1101/2024.01.07.574507

Figure Lengend Snippet: A) Principal component analysis (PCA) plot for the sequenced samples of WT, Mecp2 KO, WT+NPCs and Mecp2 KO+NPCs cerebella. Percentage of variance is reported for both PC1 (first component) and PC2 (second component). B) The number of deregulated genes (DEGs) for the different comparison is reported, considering a p.adj<0.05. The number of DEGs with a LogFoldChange lower than −1 (down-regulated genes) or LogFoldChange greater than 1 (up-regulated genes) is also indicated. C) Dot plot of Gene Ontology (GO) enriched pathway analysis in the cerebellum, indicating the top 30 most enriched pathways of the comparison between KO+NPCs versus KO samples. D) Gene set enrichment analysis (GSEA) of IFNγ response in cerebellar samples, indicating a significant enrichment of the gene set in KO+NPCs vs KO comparison, as well as in KO+NPCs vs WT comparison. E) The histogram reports the transcriptional levels of genes (Parp9, Iftm3, Irf8 and Bst2) associated to IFNγ pathway. Data, expressed as percentage of WT animals, are shown as mean ± SEM. *p<0.05, **p<0.01 by two-way ANOVA followed by Tukey post hoc test. F) Western blot analysis of phosphorylated STAT1 (P-STAT1) in WT or KO neurons cultured with NPCs (for 14 days) (n=7-9). Data are represented as mean ± SEM and expressed as percentage of WT neurons cultured alone. Representative bands of P-STAT1 and the corresponding lanes of TGX-stain free gel are reported.

Article Snippet: Then, membranes were incubated for 1 hour in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4°C with the following primary antibodies: rabbit anti-phosphorylated Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling) or rabbit anti-Bdnf (1:1000; ab108319, Abcam).

Techniques: Comparison, Western Blot, Cell Culture, Staining

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) IB analysis of phosphorylated(P)-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (B) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (C) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in cytoplasmic and nuclear extracts of HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. α-Tubulin and Histone H3 were used as the cytoplasmic and nuclear controls, respectively.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Transfection

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) ChIP analysis of STAT1/STAT2 DNA-binding in promoters of IFIT1 and IFIT2 in HeLa WT and HeLa Bclaf1-KO cells simulated with PBS or human IFNα (500U/mL) for 1h. (B) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1. Unlabeled ISRE was used for control. (C) IB analysis of ISRE-binding Bclaf1. Unlabeled ISRE and Bio-GFP were used for control. (D) ChIP analysis of Bclaf1 DNA-binding in promoters of ISG15, IFIT1 and IFIT2 in HeLa cells simulated with PBS or human IFNα (500U/mL) for 1h. An amplicon located in IFIT1 exon2 was also tested for control. (E) IB analysis of WT or mutated (1–3) Bio-ISRE pull-down Bclaf1.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Binding Assay, Amplification

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) IB analysis of STAT1, STAT2, P-STAT1, P-STAT2 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 2h. IgG was used for control immunoprecipitation. (B) IB analysis of IRF9 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 4h. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1 truncations and Ha-tagged STAT1/STAT2IRF9 expression plasmids. (D) qRT-PCR analysis of IFIT1 mRNA levels in HEp-2 cells transfected with Flag-tagged EV, full-length Bclaf1 or its truncations expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 3h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test. ***p<0.001

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Immunoprecipitation, Transfection, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) GST pulldown analysis of the interaction between His-STAT2 and GST-Bclaf1 F2. (B) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged STAT1 or STAT2/IRF9 expression plasmids. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged IRF9 or STAT2/STAT1 expression plasmids. (D) IB analysis of STAT1, STAT2, IRF9 and Flag-Bclaf1 in nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line transfected with si-control or si-STAT2 followed by PBS or human IFNα (500U/mL) treatment for 3h. (E) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Transfection, Expressing

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: Upon IFN-I binding with receptors, Bclaf1 facilitates the phosphorylation of STAT1/STAT2 in an indirect manner. Phosphorylated STAT1 and STAT2 associate with IRF9 to format a complex called ISGF3 and translocate to the nucleus. Bclaf1 is acting as a mediator attracting ISGF3 to ISGs promoters for efficient transcription. On the one hand, Bclaf1 interacts with STAT2 directly to associate with ISGF3. On the other hand, Bclaf1 binds with ISRE. During PRV or HSV-1 infection, US3 is dispatched to degrade Bclaf1 to inhibit IFN signaling.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Binding Assay, Infection

Journal: Journal of Virology

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

doi: 10.1128/JVI.00011-10

Figure Lengend Snippet: Characteristics of the RV strains and P proteins used in this study. (A) Genome organization and pathogenicities of the Nishigahara (Ni), Ni-CE, and chimeric CE(NiP) strains. The pathogenicity of each strain for adult mice was previously determined by i.c. inoculation with 1,000 FFU of each virus (30). ++, lethal (all mice died within 7 days); +, lethal (all mice died within 14 days); −, not lethal (all mice survived). (B) Amino acid differences between Nishigahara and Ni-CE P proteins are highlighted. The previously identified nuclear export signal (NES), nuclear localization signal (NLS) (20), and STAT1-binding domain (36) are also indicated. AA, amino acids. (C) Propagation of Nishigahara, Ni-CE, and CE(NiP) strains in adult mouse brains. The virus titers in the mouse brains were determined as previously described (34). †, All Nishigahara-inoculated mice died after this time point.

Article Snippet: The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red).

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

doi: 10.1128/JVI.00011-10

Figure Lengend Snippet: The subcellular localization of STAT1 in RV-infected SK-N-SH cells differs between the viral strains. (A) The cells were inoculated with each strain at an MOI of 0.01 and then were treated with IFN-α (4,000 U/ml) for 30 min at 24 h p.i. The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min before being immunostained for STAT1 (green) and RV N protein (red) and analyzed by CLSM. (B) Images such as those shown in panel A were used to calculate the ratios of nuclear to cytoplasmic fluorescence (Fn/c) of STAT1, which are shown as the means ± standard errors of the means of the results from >30 images. ns, not significant (P ≥ 0.05); Ni, Nishigahara.

Article Snippet: The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red).

Techniques: Infection, Fluorescence

Journal: Journal of Virology

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

doi: 10.1128/JVI.00011-10

Figure Lengend Snippet: The Ni-CE P protein is defective for cytoplasmic localization and for its capacity to inhibit nuclear import of IFN-activated STAT1. (A) Vero cells were transfected to express the indicated GFP-tagged P protein (green) and, 18 h later, were treated with or without IFN-α for 1 h. The cells were fixed with formaldehyde and methanol and immunostained for STAT1 (red) before being analyzed by CLSM. (B, C) Images such as those shown in panel A were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 130, combined data from 3 separate assays) for STAT1 (B) or GFP-tagged P protein (C). Ni, Nishigahara.

Article Snippet: The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red).

Techniques: Transfection, Fluorescence

Journal: Journal of Virology

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

doi: 10.1128/JVI.00011-10

Figure Lengend Snippet: Both the Nishigahara (Ni) and Ni-CE P proteins physically interact with STAT1. (A) Yeast cells (L40 strain) were cotransformed with plasmid pLex-CVS P, -Ni P, or -Ni-CE P and plasmid pGAD-STAT1 (+) or the empty pGAD plasmid (−). The P protein-STAT1 interaction was assessed by the appearance of blue colonies in the presence of X-Gal on a plate lacking Trp and Leu (upper panel) and by the expression of the His3 reporter gene on a plate lacking Trp, Leu, and His (lower panel). (B) SK-N-SH cells were inoculated with strain Ni-CE or CE(NiP) at an MOI of 0.1. At 18 h p.i., the cells were treated with IFN-α for 2 h before being lysed in RIPA buffer. The cell lysates were subjected to co-IP analysis with an anti-STAT1 antibody or control rabbit IgG. The precipitates and total lysate (input) were analyzed by Western blotting (WB). The asterisk represents an additional band probably resulting from binding of antibodies used for Western blotting to protein A/G. α-Tubulin, alpha-tubulin.

Article Snippet: The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red).

Techniques: Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Western Blot, Binding Assay

Journal: Journal of Virology

Article Title: Role of Interferon Antagonist Activity of Rabies Virus Phosphoprotein in Viral Pathogenicity ▿

doi: 10.1128/JVI.00011-10

Figure Lengend Snippet: The NES in RV P protein plays an important role in its IFN antagonism. (A) In order to restore the NES activity to the Ni-CE P protein [producing Ni-CE P(NES+)-GFP], Pro-to-Leu substitutions were introduced into Ni-CE P-GFP at positions 56 and 58. (B) To compare the subcellular localization of Ni-CE P(NES+)-GFP with that of Ni-CE P-GFP, SK-N-SH cells were transfected with plasmid pEGFP-N1 expressing the respective protein and images were collected 24 h posttransfection. (C) Vero cells were transfected to express Ni-CE P-GFP or Ni-CE P(NES+)-GFP (green) and treated with or without IFN-α before being fixed and immunostained for STAT1 (red) and analyzed by CLSM. (D, E) Images such as those shown in panel C were analyzed to derive the ratio of nuclear to cytoplasmic fluorescence (Fn/c) values (mean ± standard error of the mean, n > 30) for GFP-tagged P protein (D) or STAT1 (E). (F) SK-N-SH cells were transfected with the ISRE reporter and the control plasmids, together with the pEGFP-N1 plasmid expressing Ni-GFP, Ni-CE GFP, or Ni-CE P(NES+)-GFP. At 24 h posttransfection, the cells were treated with IFN-α (2,000 U/ml) for 6 h and the cell lysates were subjected to the dual luciferase assay. GL, firefly luciferase activity; RL, Renilla luciferase activity; ns, not significant (P ≥ 0.05).

Article Snippet: The cells were fixed with 3.7% formaldehyde for 10 min and 90% methanol for 5 min and immunostained with an anti-STAT1 rabbit antibody (sc-346; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-RV N protein mouse monoclonal antibody, followed by incubation with Alexa Fluor 488 anti-rabbit IgG (Invitrogen) (green) and Alexa Fluor 594 anti-mouse IgG (Invitrogen) (red).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Luciferase