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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The growth plate’s response to load is partially mediated by mechano-sensing via the chondrocytic primary cilium

doi: 10.1007/s00018-014-1690-4

Figure Lengend Snippet: ATDC5 chondrocytes respond to mechanical stimulation. ATDC5 cells were subjected to a 2D dynamic strain of 1 Hz and 20 % elongation for different time periods. a Actin arrangement was detected by immunocytochemistry staining with phalloidin (red). Stress fibers accumulation oriented with the strain direction is pointed with arrow. Scale bar 20μm. b Immunocytochemistry with anti phospho-p38 (green) antibody demonstrates p38 phosphorylation in respond to the mechanical stimulation. Nuclei stained with DAPI. Scale bars for DAPI and p-p38 50μm. Scale bar for the overlay 100μm c Immunocytochemistry with anti Stat1 (green) antibody showing changes in the Stat1 levels and localization with time. Scale bar 50μm. d Protein levels of p-p38, p-38, and Stat1 were quantified using western blot analysis. Cell lysates were separated on SDS-PAGE followed by bloting with antibodies against p-p38, p38, and Stat1. e Expression levels of PKD1, PKD2, IFT88, and IFT172 (F) FOS, EGR1, osteopontin (OPN), aggrecan (AGC1), were examined by real-time PCR. Values are expressed as mean ± SD of three replicates. Significantly different at P < 0.05. g SEM presentation of primary cilia on ATDC5 chondrocyte (H) Immunocytochemistry with acetylated α-tubulin (A-tb, green) and pericentrin (red) antibodies for the detection of the primary cilium, and DAPI for the nuclei (DAPI, blue)

Article Snippet: Lysates (30 µg protein) were separated by 10 % SDS-PAGE, transferred to nitrocellulose membranes, incubated overnight with primary antibody (listed bellow) at 4 °C, followed by incubation with peroxidase-conjugated secondary antibody, and detected with ECL [ 69 ]. . Antibodies for western blot Primary antibodies: rabbit polyclonal anti-p38 (c-20) (sc-535), mouse monoclonal anti-p-p38 (D-8)(SC-7973), rabbit polyclonal anti-STAT1(E-23)(sc-346), mouse monoclonal anti-GAPDH (A-3)(sc-137175) (Santa Cruz Biotechnology), rabbit polyclonal anti- KIF3A (ab11259) (abcam, Zotal), rabbit polyclonal anti-GFP (A11122) (Invitrogen).

Techniques: Immunocytochemistry, Staining, Western Blot, SDS Page, Expressing, Real-time Polymerase Chain Reaction

Journal: JCI Insight

Article Title: IL-7–dependent STAT1 activation limits homeostatic CD4 + T cell expansion

doi: 10.1172/jci.insight.96228

Figure Lengend Snippet: (A) Isolated CD4+ T cells from a healthy donor were cultured 3 days in media alone or rhIL-7 (10 ng/ml). After 3 days of culture, the cells were harvested, washed, and rested overnight to allow IL-7R reexpression (52). Rested cells were then stimulated in vitro with rhIL-7 (1 ng/ml) for 30 minutes, and cell lysates were analyzed by Western blotting with antibodies specific to p-STAT1, t-STAT1, p-STAT5, and t-STAT5. An antibody to actin was used to confirm even protein loading. Results are representative of at least 3 different donors. (B) PBMCs from healthy controls (HC, n = 22) and HIV-infected patients with viremia suppressed to < 50 copies/ml for median 17 months on cART (HIV+, n = 53) were analyzed for t-STAT1 and p-STAT1 levels in total CD4+ and CD8+ T cell populations. The relationship between the STAT1 phosphorylation after 30 minutes in vitro stimulation with rhIL-7 and t-STAT1 levels was assessed using a nonparametric Spearman test.

Article Snippet: Antibodies to STAT1 (catalog sc-346) and STAT5 (catalog sc-835) were from Santa Cruz Biotechnology Inc. Phosphorylation-specific antibodies to STAT1 (Tyr701; catalog 9171) and STAT5 (Tyr694; catalog 9351) were from Cell Signaling Technology.

Techniques: Isolation, Cell Culture, In Vitro, Western Blot, Infection, Phospho-proteomics

Journal: JCI Insight

Article Title: IL-7–dependent STAT1 activation limits homeostatic CD4 + T cell expansion

doi: 10.1172/jci.insight.96228

Figure Lengend Snippet: Lymphoreplete B6 CD45.1 (B6 host, n = 7) and lymphopenic RAG–/– CD45.1 (RAG–/– host, n = 11) mice were injected i.v. with 10 × 106 of CellTrace Violet–labeled (CTV-labeled) lymph node (LN) cells from congenic B6 CD45.2 mice. Analysis of transferred cells was performed on day 7 after transfer. The expression levels of STAT1 and activated p-STAT1 and p-STAT5 of donor T cells were evaluated by flow cytometry in LNs as function of CTV fluorescence after in vitro stimulation with rmIL-7 (1 ng/ml). Donor T cells undergoing slow proliferation (SP, CTV+ cells gated in blue) and fast proliferation (FP, CTV– cells gated in green) after transfer into RAG–/– hosts were analyzed separately. (A) The percentages of donor T cells CTV+ t-STAT1high, CTV– t-STAT1high, and CTV– t-STAT1low are indicated. The MFIs of t-STAT1 in CD4+ and CD8+ donor T cells 7 days after adoptive transfer into lymphoreplete B6 (black symbols) and lymphopenic RAG–/– hosts undergoing SP (blue symbols) or FP (green symbols) were compared using a nonparametric Mann-Whitney test. (B) The percentages of donor T cells CTV+ p-STAT1high, CTV+ p-STAT1low, CTV– p-STAT1high, and CTV– p-STAT1low and CTV+ p-STAT5high, CTV+ p-STAT5low, CTV– p-STAT5high, and CTV– p-STAT5low are indicated. The MFIs of p-STAT1 and p-STAT5 in CD4+ and CD8+ donor T cells after adoptive transfer into lymphoreplete B6 (black symbols) and lymphopenic RAG–/– hosts undergoing SP (blue symbols) or FP (green symbols) were compared using a nonparametric Mann-Whitney test. Data are presented as box and whisker plots showing the median MFI value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values, from 3 independent experiments out of 3, including an average of 2–4 mice per group per experiment.

Article Snippet: Antibodies to STAT1 (catalog sc-346) and STAT5 (catalog sc-835) were from Santa Cruz Biotechnology Inc. Phosphorylation-specific antibodies to STAT1 (Tyr701; catalog 9171) and STAT5 (Tyr694; catalog 9351) were from Cell Signaling Technology.

Techniques: Injection, Labeling, Expressing, Flow Cytometry, Fluorescence, In Vitro, Adoptive Transfer Assay, MANN-WHITNEY, Whisker Assay

Journal: JCI Insight

Article Title: IL-7–dependent STAT1 activation limits homeostatic CD4 + T cell expansion

doi: 10.1172/jci.insight.96228

Figure Lengend Snippet: LN cells from WT (n = 10) and STAT1 Tg (n = 10) mice were analyzed by flow cytometry for expression of (A) t-STAT1 and (B) phosphorylated STAT1 and STAT5 after in vitro stimulation with rmIL-7 (1 ng/ml) in B cells (CD3– B220+), CD4+ (B220– CD3+ CD4+), and CD8+ (B220– CD3+ CD8+) T cells. Shaded histograms represent isotype control staining. The MFIs of t-STAT1, p-STAT1, and p-STAT5 in the different populations were compared between WT (black symbols) and STAT1 Tg (red symbols) mice using a nonparametric Mann-Whitney test. Data from 3 independent experiments, including an average of 3–4 mice per group per experiment, are presented as box and whisker plots showing the median MFI value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values. (C) Gene set enrichment analysis (GSEA) histograms of IFN signature in STAT1 Tg versus WT CD4+ and CD8+ naive T cells stimulated with IL-7. The enrichment score (ES; y axis) reflects the degree to which IFN signature was enriched in STAT1 Tg vs. WT T cells. Each solid bar represents 1 gene within IFN signature. The heat-map image illustrates the gene expression levels of the leading edge subset. The normalized enrichment score (NES) and false discovery rate (FDR) q value are indicated.

Article Snippet: Antibodies to STAT1 (catalog sc-346) and STAT5 (catalog sc-835) were from Santa Cruz Biotechnology Inc. Phosphorylation-specific antibodies to STAT1 (Tyr701; catalog 9171) and STAT5 (Tyr694; catalog 9351) were from Cell Signaling Technology.

Techniques: Flow Cytometry, Expressing, In Vitro, Control, Staining, MANN-WHITNEY, Whisker Assay, Gene Expression

Journal: JCI Insight

Article Title: IL-7–dependent STAT1 activation limits homeostatic CD4 + T cell expansion

doi: 10.1172/jci.insight.96228

Figure Lengend Snippet: Lymphopenic RAG–/– mice were injected i.v. with 6 × 106 of CTV-labeled T cells isolated from the LNs of congenic WT or STAT1 Tg mice. Analysis of donor cells in LNs was performed on day 7 after transfer. (A) Percentages of slow and fast proliferating (SP, CTV+ cells gated in blue; FP, CTV– cells gated in green) CD45.2+ CD3+ CD4+ and CD8+ donor lymphocytes are shown. (B) The percentages of donor T cells CTV+ t-STAT1high, CTV– t-STAT1high, and CTV– t-STAT1low are indicated. The MFIs of t-STAT1 in CD4+ and CD8+ donor T cells before (d0) and after adoptive transfer (SP and FP) with WT (n = 13, black symbols) or STAT1 Tg (n = 15, red symbols) were compared using a nonparametric Mann-Whitney test. (C) The percentages of donor T cells CTV+ p-STAT1high, CTV+ p-STAT1low, CTV– p-STAT1high, and CTV– p-STAT1low are indicated. The MFIs of p-STAT1 in CD4+ and CD8+ donor T cells before (d0) and after adoptive transfer into RAG–/– mice (SP) were compared as described above. Data are from 4 independent experiments out of 4, including an average of 3–4 mice per group per experiment and presented as box and whisker plots showing the median value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values.

Article Snippet: Antibodies to STAT1 (catalog sc-346) and STAT5 (catalog sc-835) were from Santa Cruz Biotechnology Inc. Phosphorylation-specific antibodies to STAT1 (Tyr701; catalog 9171) and STAT5 (Tyr694; catalog 9351) were from Cell Signaling Technology.

Techniques: Injection, Labeling, Isolation, Adoptive Transfer Assay, MANN-WHITNEY, Whisker Assay

Journal: JCI Insight

Article Title: IL-7–dependent STAT1 activation limits homeostatic CD4 + T cell expansion

doi: 10.1172/jci.insight.96228

Figure Lengend Snippet: Lymphopenic RAG–/– mice transferred with WT (n = 16) or STAT1 Tg (n = 15) T cells were analyzed as described above. (A) Absolute numbers of CD4+ and CD8+ donor T cells were enumerated following T cell transfer with WT (black symbols) or STAT1 Tg (red symbols) cells. (B) Expression of CD44 and CD62L on CD4+ and CD8+ donor T cells was assessed by flow cytometry in LNs. The percentages and absolute cell counts of CD4+ and CD8+ donor T cell subsets in RAG–/– mice transferred with WT (black symbols) or STAT1 Tg (red symbols) T cells are presented. (C) LN cells were stimulated ex vivo with PMA/ionomycin. IFN-γ production by donor T cells was assessed by intracellular staining, and plots represent CFSE fluorescence versus IFN-γ on gated CD45.2+ CD3+ CD4+ and CD8+ lymphocytes. The percentages of IFN-γ–producing donor T cells are indicated. Background in nonstimulated controls was < 1%. A nonparametric Mann-Whitney test was performed for comparisons between groups. Data are from 5 independent experiments out of 5, including an average of 3–4 mice per group per experiment and presented as box and whisker plots showing the median value bounded by the first and third quartiles in the box, with whiskers extending to the minimum and maximum values.

Article Snippet: Antibodies to STAT1 (catalog sc-346) and STAT5 (catalog sc-835) were from Santa Cruz Biotechnology Inc. Phosphorylation-specific antibodies to STAT1 (Tyr701; catalog 9171) and STAT5 (Tyr694; catalog 9351) were from Cell Signaling Technology.

Techniques: Expressing, Flow Cytometry, Ex Vivo, Staining, Fluorescence, MANN-WHITNEY, Whisker Assay