Journal: Molecular Metabolism

Article Title: Baricitinib counteracts metaflammation, thus protecting against diet-induced metabolic abnormalities in mice

doi: 10.1016/j.molmet.2020.101009

Figure Lengend Snippet: Baricitinib attenuates the HD-induced JAK-STAT pathway. Western blotting analysis for phosphorylation of Tyr 1007/1008 JAK1/2 in the skeletal muscle (A) and kidneys (B) and normalized to total JAK1/2 and for phosphorylation of Tyr 690 on STAT2 in the skeletal muscle (C) and kidneys (D) and normalized to total STAT2. All of the data are expressed as mean ± SEM for n = 6 per group. ∗p< 0.05 vs ND and •p< 0.05 vs HD.

Article Snippet: The antibodies used were rabbit anti-Tyr 1007/1008 JAK2 (#3776), rabbit anti-total JAK2 (#3230), rabbit anti-Tyr 690 STAT2 (Bioss Antibodies, bs-3428R), rabbit anti-total STAT2 (#72604), rabbit p21 (#2947), rabbit anti-Ser 307 IRS-1 (#2381), mouse anti-total IRS-1 (#3194), rabbit anti-Ser 473 AKT (#4060), rabbit anti-total AKT (#9272), rabbit anti-Ser 9 GSK-3β (#9332) and rabbit anti-total GSK-3β (9315).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Influenza A Virus Induces Interleukin-27 through Cyclooxygenase-2 and Protein Kinase A Signaling

doi: 10.1074/jbc.m111.308064

Figure Lengend Snippet: FIGURE 6. IL-27 treatment stimulates STAT1, STAT2, STAT3, and PKR phosphorylation. A549 cells were treated with recombinant IL-27 (50 ng/ml) for 12 h. Cells were harvested for the detection of: A, STAT1 and phosphorylated STAT1; B, STAT2 and phosphorylated STAT2; C, STAT3 and phosphorylated STAT3; or D, PKR and phosphorylated PKR expression. Densitometric analysis was normalized to the corresponding control in each experiment (*, p 0.05). E, IFN- expression level in IL-27-treated PBMCs. For IFN- neutralization and antiviral assays, freshly isolated PBMCs were treated with recombinant IL-27 (100 ng/ml) for 2 h, then the culture medium supernatant was harvested and incubated with IFN- neutralization antibody (5 l/ml) or control IgG (5 l/ml) at 37 °C for 1 h. The IFN- Ab-neutralized culture medium was then used for the following antiviral assays (*, p 0.05). F, A549 cells were incubated with IFN- Ab-neutralized or control IgG-neutralized culture medium for 12 h before IAV A/Hong Kong/498/97 (H3N2) infection (m.o.i. 1). Cells were collected 3 h post-infection and total RNA was isolated for detection of NP-specific mRNA, cRNA, and vRNA. The values of IAV-infected A549 cells for three RNAs were designated as 1. Data are expressed as fold-induction relative to the values of IAV-infected A549 cells for three RNAs (*, p 0.05). G and H, A549 cells were treated as described in F for 12 h, then phosphorylated STAT1 (G) and phosphorylated PKR (H) were detected. The blot is representative of three experiments with similar results. Densi- tometric analysis relative to STAT1 or PKR levels was expressed as fold-change (*, p 0.05).

Article Snippet: A polyclonal rabbit antibody specific for STAT1 (9172) and a polyclonal rabbit antibody specific for phosphorylated STAT2 (4441) and phosphorylated STAT3 (9145) were purchased from Cell Signaling (Danvers, MA).

Techniques: Phospho-proteomics, Recombinant, Expressing, Control, Neutralization, Isolation, Incubation, Infection

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of EHV-1 infection on IFN-induced STAT1/STAT2 phosphorylation. EECs were either mock infected (M) or infected with T953 (E1) at an MOI of 5, and cells were either treated with equine rEqIFN-α at 1,000 IU/ml or left untreated (+ or −, respectively) for 30 min before lysis. Abundance of phosphorylated versus total STAT1 at 12 h (A) and phosphorylated versus total STAT2 at 3 h (B), 6 h (C), or 12 h (D) were then quantified using Western blot analysis. Membranes were stripped and reprobed with β-actin as a control for equal loading of samples. Densitometric analysis of Western blot images for phosphorylated STAT1 and total STAT1 (E) and phosphorylated STAT2 and total STAT2 (F) normalized against β-actin was conducted using ImageJ (NIH). The Western blot images are representative of 3 different blots from independent experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Lysis, Western Blot

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of EHV-1 infection on nuclear accumulation of STAT2. EECs grown on coverslips in 24-well plates were either infected with T953 at an MOI of 3 or mock infected. The cells were either stimulated with equine rEqIFN-α at 1,000 IU/ml or treated with an equal volume of plain medium for 30 min prior to fixation in 4% PFA at 12 hpi. Cells were stained with anti-STAT2 (1:100 dilution) and anti-EHV-1 gC antibody (1:200 dilution). Approximately 300 cells from different fields were examined for each treatment using an inverted fluorescence microscope. (A) Mock-infected EECs treated with plain medium and stained for STAT2. (B) Mock-infected cells stimulated with equine rEqIFN-α and stained for STAT2. (C) T953-infected cells stained for STAT2. (D) T953-infected cells stimulated with equine rEqIFN-α and stained for STAT2. 4,6-Diamidino-2-phenylindole (DAPI; blue) served as a counterstain. Solid arrows indicate representative cells without nuclear STAT2 translocation, whereas open arrows indicate representative cells with nuclear STAT2 translocation. Scale bars, 50 μm. (E) Quantification of nuclear STAT2 intensity of IF images using Nikon NIS-Elements software as described in Materials and Methods. (F) EECs were either mock infected (M) or infected with T953 (E1) at an MOI of 3, and cells were either treated with equine rEqIFN-α (1,000 IU/ml) or left untreated (+ or −, respectively) for 30 min before fractionation into nuclear (N) or cytoplasmic (C) compartments. Separated cellular fractions were probed for phosphorylated STAT2 and total STAT2 to determine their cellular distribution following T953 infection. Lamin A/C and MEK1/2 were used as nuclear and cytoplasmic loading controls, respectively.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Staining, Fluorescence, Microscopy, Translocation Assay, Software, Fractionation

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of UV inactivation on type I IFN molecules. Mock-infected EECs (M, solid bars) were treated with either 80 μg/ml of poly(I·C) (A, C, and D) or 10 μg/ml of LPS (B) (both labeled as P, white bars) or infected with UV-inactivated T953 in the absence (E1, checkerboard bars) or presence (P+E1, diagonally striped bars) of P as described in the text. At the indicated time points, the cells were lysed and equine TLR3 (A), TLR4 (B), IRF7 (C), and IRF9 mRNA (D) were quantified by real-time RT-PCR. Data were normalized to the levels of endogenous control equine RPLP0 mRNA at the same time point. ns, not significant. (E) EECs were either mock infected (M) or infected with UV-inactivated T953 (E1) at an MOI of 5, and cells were either treated with 1,000 IU/ml of equine rEqIFN-α or left untreated (+ or −, respectively) for 30 min prior to lysis. At 12 hpi, cells were lysed, and equal amounts of proteins were separated on 10% SDS-PAGE and probed using phospho-STAT2 or STAT2. Images are representative of 3 independent biological experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Infection, Labeling, Quantitative RT-PCR, Lysis, SDS Page

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Effect of viral late gene blockage on STAT2 activation. EECs were infected with T953 (E1) either in the presence (+) or absence (−) of PAA (300 μg/ml). Mock-infected EECs (M) were either treated with equine rEqIFN-α at 1,000 IU/ml or left untreated (+ or −, respectively) for 30 min before lysis. Abundance of phosphorylated versus total STAT2 at 3 h (A), 6 h (B), or 12 h (C) was then quantified using Western blot analysis. Membranes were stripped and reprobed with β-actin as a control for equal loading of samples. As controls for effective PAA-mediated blockage of T953 L proteins, both the EHV-1 IE and EHV-1 gD were also quantified. The Western blot images are representative of 3 different blots from independent experiments.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Activation Assay, Infection, Lysis, Western Blot

Journal: Journal of Virology

Article Title: Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells

doi: 10.1128/JVI.01342-19

Figure Lengend Snippet: Schematic illustration of our proposal of how EHV-1 T953 strain blocks the key molecules required for type I IFN production. (A) During the sensitization phase of host type I IFN production, EHV-1 blocks the expression of TLR3 and TLR4 mRNA, thereby enabling the virus to avoid host detection. (B) In the subsequent induction phase, EHV-1 specifically degrades the cellular level of TYK2 protein. This consequently blocks the abundance of cellular levels of phosphorylated/activated STAT1 and STAT2 molecules in infected cells. At the same time, EHV-1 blocks the transcription of IRF9 mRNA and thus prevents the formation of ISGF3 complex needed to transactivate ISRE for ISG production. EHV-1 also inhibits the transcription of IRF7 mRNA, exerting a negative effect on IFN-α production. The overall effect of EHV-1 renders the host cell unable to restrict viral spread by direct cell-to-cell contact during infection.

Article Snippet: Rabbit anti-phospho-STAT2 primary antibody was purchased from Rockland Immunochemicals Inc. (Limerick, PA).

Techniques: Expressing, Infection

Journal: Frontiers in microbiology

Article Title: African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

doi: 10.3389/fmicb.2023.1169699

Figure Lengend Snippet: FIGURE 2 ASFV pA104R inhibits ISGF3-induced ISRE promoter activity and attenuates the phosphorylation of STAT1. (A) HEK-293 T cells were transfected with various concentrations of pA104R plasmids, along with ISGF3 complex (STAT1, STAT2 and IRF9) and pISRE-Luc and pRL- TK plasmids. After 30 h, a luciferase assay was performed. Data are shown as means ± SEM from three independent experiments. Statistical analysis was performed by one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. (B) HEK-293 T cells were transfected with pA104R or empty vector. After 24 h, cells were treated with 1,000 U/ ml IFN-α for 2 h. The levels of total or phosphorylated STAT1, STAT2, and IRF9 were detected by immunoblotting analysis.

Article Snippet: The STAT1 (9172), STAT2 (72604), phosphor-STAT1 (9649), phosphor-STAT2 (88410), and IRF9 (76684) antibodies were purchased from Cell Signaling Technology (Danvers, MA). β-Actin (66009-1-Ig), GFP-tag (66002-1-Ig), Flag-tag (20543-1-AP), and HA-tag (51064-2-AP) antibodies were purchased from Proteintech (Chicago, IL).

Techniques: Activity Assay, Phospho-proteomics, Transfection, Luciferase, Plasmid Preparation, Western Blot

Journal: Frontiers in microbiology

Article Title: African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

doi: 10.3389/fmicb.2023.1169699

Figure Lengend Snippet: FIGURE 3 ASFV pA104R does not interact with STAT1, STAT2, and IRF9. HEK-293 T cells were transfected with pA104R alone (C) or co-transfected with STAT1, STAT2, and IRF9 (A,B). After 24 h, cells were treated with 1,000 U/mL IFN-α for 8 h. Cell lysates were prepared and subjected to immunoprecipitation analysis. The whole-cell lysates and immunoprecipitation complexes were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: The STAT1 (9172), STAT2 (72604), phosphor-STAT1 (9649), phosphor-STAT2 (88410), and IRF9 (76684) antibodies were purchased from Cell Signaling Technology (Danvers, MA). β-Actin (66009-1-Ig), GFP-tag (66002-1-Ig), Flag-tag (20543-1-AP), and HA-tag (51064-2-AP) antibodies were purchased from Proteintech (Chicago, IL).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Frontiers in microbiology

Article Title: African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

doi: 10.3389/fmicb.2023.1169699

Figure Lengend Snippet: FIGURE 5 ASFV pA104R does not prevent the association of ISGF3 with promoter. (A) The ISRE DNA and control oligonucleotides used for DNA pull-down. (B) HEK-293T cells were co-transfected with ISGF3 complex (STAT1, STAT2, and IRF9), along with pA104R or empty control. After 24 h post- transfection, cells were treated with 1,000 U/mL IFN-α for 12 h. Nuclear extracts were incubated with a Biotin-labeled ISRE or control probe and subjected to pull-down analysis with streptavidin magnetic beads. The whole-cell lysates and pull-down complexes were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: The STAT1 (9172), STAT2 (72604), phosphor-STAT1 (9649), phosphor-STAT2 (88410), and IRF9 (76684) antibodies were purchased from Cell Signaling Technology (Danvers, MA). β-Actin (66009-1-Ig), GFP-tag (66002-1-Ig), Flag-tag (20543-1-AP), and HA-tag (51064-2-AP) antibodies were purchased from Proteintech (Chicago, IL).

Techniques: Control, Transfection, Incubation, Labeling, Magnetic Beads, Western Blot

Journal: Frontiers in microbiology

Article Title: African swine fever virus pA104R protein acts as a suppressor of type I interferon signaling.

doi: 10.3389/fmicb.2023.1169699

Figure Lengend Snippet: FIGURE 6 ASFV pA104R does not exert repressive effects on trans-activation domains and transcriptional co-stimulatory factors. (A) HEK-293 T cells were co- transfected with pA104R and IRF9-Stat2(TA) (A) or GAL4-Stat2(TA) (B), along with pRL-TK and pISRE-Luc (A) or pGAL4-UAS-Luc (B) plasmids. After 30 h, a luciferase assay was performed. (C) HEK-293 T Cells were co-transfected with pA104R and CBP or p300 along with pISRE-Luc and pRL-TK plasmids. After 24 h post-transfection, cells were treated with 1,000 U/mL IFN-α for 12 h, followed by luciferase assays. Data are shown as means ± SEM from three independent experiments. Statistical analysis was performed by one-way ANOVA. *** p < 0.001.

Article Snippet: The STAT1 (9172), STAT2 (72604), phosphor-STAT1 (9649), phosphor-STAT2 (88410), and IRF9 (76684) antibodies were purchased from Cell Signaling Technology (Danvers, MA). β-Actin (66009-1-Ig), GFP-tag (66002-1-Ig), Flag-tag (20543-1-AP), and HA-tag (51064-2-AP) antibodies were purchased from Proteintech (Chicago, IL).

Techniques: Activation Assay, Transfection, Luciferase

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) TRExBCBL1-RTA (iBCBL-1) cells, doxycycline (Dox)-inducible for viral immediate-early RTA expression , were either left untreated (latently infected) or treated with Dox (1 μg/ml) for 1 day to induce lytic replication. Cells were harvested and lysed for the preparation of whole-cell extracts and these were used for immunoprecipitation (IP) of IRF9, STAT1, or STAT2; for IRF9, two different IP antibodies (Ab-1, Ab-2) were used. Non-immune mouse IgG was used as a negative control. Immunoprecipitates and cell lysates were immunoblotted for detection of IRF9, STAT1, STAT2, and vIRF-1. The immunoprecipitated vIRF-1 bands, running close to unblocked Ig heavy chain (above), are indicated by arrowheads. The dotted line indicates deletion of lanes, from a single blot. (B) Colocalization of vIRF-1 with STAT1 and STAT2 was investigated by proximity ligation assay (PLA). Rabbit antiserum to vIRF-1 was paired with either rat (STAT1) or mouse (STAT2) antibody, and appropriate detection antibodies were used for PLA (see ). Negative controls comprised non-specific mouse IgG (IgG m ) or rabbit IgG (IgG r ) coupled with vIRF-1 or STAT antibodies, respectively. Cells were visualized by confocal microscopy to detect fluorescent dots, indicative of the colocalizations of complementary species-specific antibodies and therefore of the corresponding vIRF-1 and STAT targets. Untreated (latent) or Dox-induced (lytic) iBCBL-1 cells were analyzed; lytic cultures were harvested 1 day after induction. (C-E) In vitro coprecipitation assays using bacterially-expressed and purified STAT1, STAT2, and IRF9 (Flag-tagged) along with S-tagged and affinity-precipitated vIRF-1. Coprecipitated target proteins were detected by Flag immunoblotting, and vIRF-1-S affinity-precipitation (AP) was verified by S-peptide antibody probing (arrowheads indicate vIRF-1-S; asterisks indicate remnant Flag signal from STAT1/2 degradation products or full-length IRF9 on S-blots, following sequential probing). The input proteins used in each assay were quality-checked on Ponceau-S-stained gels (bottom panels).

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Expressing, Infection, Immunoprecipitation, Negative Control, Proximity Ligation Assay, Confocal Microscopy, In Vitro, Purification, Western Blot, Affinity Precipitation, Staining

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) Analysis of phosphorylated (active) STAT1 and STAT2 (pSTAT1, pSTAT2) and total STAT1 and STAT2 in latent (-Dox) and lytic (+Dox, 2 days) iBCBL-1 PEL cells, transduced with lentivirus vector expressing either non-silencing control (ns) or vIRF-1 mRNA-specific (virf1) shRNA. Cultures remained untreated (mock) or were treated with IFNβ (50 ng/ml). Derived cell extracts were immunoblotted for assessments of pSTAT1 and pSTAT2 levels, in addition to validation of vIRF-1 induction (+Dox) and depletion and monitoring of IRF9 expression. Quantified pSTAT1 (pS1) and pSTAT2 (pS2) levels for IFNβ-treated samples, normalized to total STAT1 (S1) and STAT2 (S2), respectively, and expressed as ratios of levels in vIRF-1 versus control shRNA-transduced cells (virf1/ns), are shown below the blots. Dotted lines indicate rearrangement (for consistency) and deletion of lanes. (B) Analysis of STAT1 and STAT2 regulation as a function of vIRF-1 in BAC16 HHV-8-infected iSLK cells. Cultures infected with wild-type (wt) or vIRF-1-knockout (ttg) virus were treated with Dox and sodium butyrate (NaB) to induce lytic replication or left untreated (latent infection), with or without IFNβ treatment. Cells were harvested 3 days after treatment, and cell lysates were analyzed as above. Levels of pSTAT1 and pSTAT2, normalized to total STAT1 and STAT2, in ttg-virus-infected cells relative to wt-virus-infected cells are indicated below the blots. Arrowheads indicate positions of pSTAT1 and vIRF-1 bands; asterisks indicates non-specific bands. For A and B: nd, not determined; -, undetectable pSTAT. (C) Detection of lytic cycle-regulated ISG15 and ISG56 in iBCBL-1 cells. Expression of representative ISGs, ISG15 and ISG56, and also IFNβ, were assessed in latent and (1-day) lytic cells by RT-qPCR analysis of the respective transcripts. Data are from biological triplicates; error bars represent standard deviations from the means, and student t-test P values (two-tailed) are shown. (D) ISG15 and ISG56 transcript levels were assessed in lytically-infected iBCBL-1 cells 2 days post-Dox treatment, with or without vIRF-1 depletion, in the presence (IFNβ) or absence (mock) of IFNβ treatment (50 ng/ml). RT-qPCR for vIRF-1 mRNA confirmed vIRF-1 depletion in vIRF-1 (virf1) shRNA-expressing relative to non-silencing (ns) shRNA-expressing cells (left). (E) Equivalent analysis of ISG15 and ISG56 expression in iSLK cells infected with wild-type (wt) or vIRF-1-knockout (ttg) BAC16 virus and lytically reactivated with Dox/NaB treatment for 3 days.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Transduction, Plasmid Preparation, Expressing, Control, shRNA, Derivative Assay, Biomarker Discovery, Infection, Knock-Out, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) 293T cells transfected with empty vector (-) or vIRF-1 expression plasmid (virf1) were left untreated or treated with IFNβ or IFNγ (50 ng/ml of each) for 24 h. Cells were then harvested and lysates analyzed by immunoblotting for relative levels of pSTAT1 and pSTAT2, total STAT1 and STAT2, and IRF9. As applicable (detected), pSTAT1/STAT1 and pSTAT2/STAT2 ratios (“empty vector” values set at 1) are shown below the respective blots for IFNβ- and IFNγ-treated cells. (B) vIRF-1 suppression of IFNβ-induced signaling, as measured by ISRE-luciferase (luc) reporter assay, was detected in 293T cells cotransfected with the reporter and empty vector (-) or expression plasmid for vIRF-1 (virf1), left untreated or treated with IFNβ (50 ng/ml) for 24 h. Relative luciferase assay-derived luminescence values (RLU, means from duplicate transfectants) are shown along with standard deviations from the means. Student’s t-test P value (unpaired, two-tailed) for vIRF-1 suppression of IFNβ signaling (vIRF-1-vector relative to empty-vector RLU) is 0.0005. (C) STAT1, STAT2, and IRF9 knockout (KO) 293T cell lines were generated by Cas9/gRNA transduction, blasticidin selection, and clonal isolation of cells (see ). Target-gene ablation in each of the cell lines was tested functionally by ISRE-luciferase reporter assay following IFNβ stimulation (chart; N = 2). Mock, untreated cultures; virf1, vIRF-1 vector-transfected cells; -, empty vector-transfected cells. (D) vIRF-1 suppression of IFNβ-activated STAT1 and STAT2 (pSTAT1, pSTAT2) as a function of STAT1, STAT2, and IRF9 knockout. Cultures were transfected with empty vector (-) or vIRF-1 expression plasmid (virf1) and, after 6 h, treated with IFNβ (50 μg/ml) for 24 h. Cell lysates were analyzed by immunoblotting; numbers below the pSTAT1 and pSTAT2 blots show quantified pSTAT1 and pSTAT2 levels normalized to total STAT1 and STAT2, respectively, in the absence (value set at 1) and presence of vIRF-1. Arrows, STAT1/pSTAT1; asterisks, non-specific bands; dotted lines, lane deletions from single blot; solid line, different membrane.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase, Reporter Assay, Derivative Assay, Two Tailed Test, Knock-Out, Generated, Transduction, Selection, Isolation, Membrane

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) 293T cells transfected with empty vector (control) or vIRF-1 expression plasmid were left untreated (0 h) or treated with IFNβ (50 ng/ml) for different times (0.25 to 8 h). Cells were harvested, lysed, and analyzed by immunoblotting for detection of phosphorylated and total STAT1 and STAT2, IRF9, and also vIRF-1, to check expression in the transfectants. Relative levels of pSTAT1 and pSTAT2, normalized to STAT1 and STAT2, respectively, and with empty vector (vec) values at 0.25 h set at 1, are shown in the adjacent charts. (B) An analogous experiment was performed in STAT2-knockout (KO) 293T cells. The immunoblot-derived pSTAT1 levels, normalized to STAT1 and expressed relative to vector/0.25 h pSTAT1 (set at 1), are plotted in the chart (right). For both panels, dotted lines indicate lane deletion or rearrangement of blot sections for consistency of presentation.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Knock-Out, Derivative Assay

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A-C) Flag-tagged STAT1 (A) or IRF9 (C) or CBD-tagged STAT2 (B) were expressed in 293T cells transfected with the respective expression plasmids and either vIRF-1 (virf1) or empty (-) expression vector; replicates were either left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h. Flag/CBD-tagged proteins were then immuno/affinity-precipitated from cell lysates, and coprecipitated ISGF3 proteins were detected by immunoblotting. Diagrams below the panels illustrate the main findings from each experiment. (D-E) Serial coprecipitations of STAT1 and STAT2 (D), STAT1 and IRF9 (E), and IRF9 and STAT2 (F), respectively Flag- and CBD-tagged, from lysates of transfected 293T cells expressing vIRF-1 (virf1) or cotransfected with empty vector (-). All transfectants were treated with IFNβ for 24 h prior to harvesting. First (Flag IP) and second (CBD AP) precipitates (Precip. 1, Precip. 2) were analyzed by immunoblotting for the tagged “bait” proteins, the third (endogenous) ISGF3 protein (including phosphorylated and total STAT1 and STAT2), and vIRF-1; lysates were immunoblotted for expression of input proteins. Illustrated below each panel of blots are the main findings. (G) Disruption by vIRF-1 of STAT1-STAT2 complexes isolated by Flag-IP (STAT1) and CBD-AP (STAT2) from IFNβ-treated transfected 293T cells. Immunoprecipitated material from vIRF-1-Flag (virf1) or empty control (cntl) vector-transfected cells was applied in two concentrations (1x, 2x) to dual-precipitation-derived STAT1/STAT2 complexes, and then mixtures were subjected to re-precipitation with chitin beads (binding STAT2-CBD). STAT1 and vIRF-1 associated with re-precipitated STAT2-CBD were identified by immunoblotting. Relative levels of co-precipitated STAT1, normalized to affinity-sedimented STAT2, are shown below the STAT1 blot (cntl/1x value set at 1). (H) An equivalent experiment was carried out using GST-fused recombinant vIRF-1 (virf1) or GST (negative control) to challenge STAT1 interaction with STAT2 in STAT1/STAT2 hetero-complexes isolated by IP/AP dual precipitations from IFNβ-treated 293T cells. Endogenous IRF9 interaction with STAT1/2 and competition by vIRF-1 were also monitored. Relative levels and integrities of the recombinant proteins are shown in the Coomassie-stained gel (right); arrowheads indicate the positions of the full-length proteins.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Disruption, Isolation, Immunoprecipitation, Control, Derivative Assay, Binding Assay, Recombinant, Negative Control, Staining

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: (A) TYK2-S and STAT2-CBD were expressed with (virf1) or without (-) vIRF-1 in transfected 293T cells. Cell lysates and S-protein affinity-precipitates were assessed for input protein expression and sedimentation by immunoblotting with CBD (STAT2), vIRF-1, and S-tag (TYK2) antibodies. Affinity-precipitated TYK-2-S was probed with phospho-tyrosine (PY)-specific antibody to identify the active, autophosphorylated form of the kinase. Dotted lines indicate lane deletions from single membranes; the arrowhead and asterisk indicate CBD-specific (STAT2) band and remnant S-tag signal (after blot stripping), respectively. (B) An equivalent experiment was performed to assess vIRF-1 effects on JAK1 autophosphorylation and association with STAT2. Here, STAT2 antibody was used to detect endogenous protein. Arrowheads indicate JAK1-S (~130 kDa). (C) ISRE-luciferase reporter assay to assess vIRF-1 inhibition of TYK2-mediated signal transduction in 293T cells cotransfected with TYK2-expression and reporter plasmids and either vIRF-1 (virf1) or empty (-) expression vectors. Average values from duplicate samples for each condition are shown; error bars indicate standard deviations from the means. Statistical significance (P) was determined by student t-test (two-tailed, unpaired). (D) IFNAR1-S-based coprecipitation assay to test the influence of vIRF-1 (virf1), relative to empty-vector (-) transfection, on STAT2-receptor association, following IFNβ stimulation for 30 min. STAT2-CBD vector cotransfection provided expression of STAT2 above endogenous levels, to facilitate detection. (E) Effect of vIRF-1 on IFNβ receptor (IFNAR1) activation and association with TYK2. Transfectants expressing vIRF-1 or containing empty vector (-, negative control) and expressing, or lacking (-), introduced IFNAR1-CBD were left untreated (mock) or treated with IFNβ (10 ng/ml) for 24 h; TYK2-S was expressed in a subset of the transfected cultures. Cell lysates were analyzed for expression of the introduced proteins, and IFNAR1-CBD was affinity-precipitated from a subset of lysates to assess interaction of the receptor with TYK2 in response to vIRF-1. The numbers below the CBD blots show relative levels (-/+ vIRF-1) of IFNβ-induced lower IFNAR1 band (arrowheads) to total IFNAR1 (top plus bottom bands) from TYK2-overexpressing transfectants (+TYK2) and those devoid of TYK2 expression plasmid (-TYK2); values in the absence (-) of vIRF-1 are set at 1. For all precipitations (panels A, B, D and E), cultures were treated with DSP (2 mM, 30 min.) immediately prior to cell harvest, to stabilize targeted complexes.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Transfection, Expressing, Sedimentation, Western Blot, Stripping, Luciferase, Reporter Assay, Inhibition, Transduction, Two Tailed Test, Plasmid Preparation, Cotransfection, Activation Assay, Negative Control

Journal: PLoS Pathogens

Article Title: STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling

doi: 10.1371/journal.ppat.1010676

Figure Lengend Snippet: IFNβ binds to IFNAR1 and IFNAR2 to effect receptor activation through auto/cross-phosphorylation of receptor associated JAK kinases (JAK1 and TYK2) and receptor tyrosine residues. STAT1 and STAT2 are recruited and activated through JAK-mediated tyrosine phosphorylation, and then can heterodimerize and associate with IRF9 to form nuclear-localizing and transcriptionally active ISGF3 complexes. Data presented here show that vIRF-1 binds directly to STAT1, STAT2 and IRF9 and can also associate, directly or indirectly, with TYK2 and JAK1. vIRF-1 inhibits TYK2, but not JAK1, autophosphorylation (activation), likely contributing to pSTAT2 suppression; vIRF-1 also inhibits TYK2-STAT2 and TYK2-IFNAR1 association (not illustrated). However, suppression of pSTAT1 is mediated largely after IFNβ-induced STAT1 phosphorylation, involving increased rates of pSTAT1 decay, likely via phosphatase (P’ase)-mediated dephosphorylation (rather than pSTAT1 degradation). Suppression of pSTAT1 by vIRF-1 is dependent on STAT2, being negated by STAT2 knockout in 293T cells; this along with decreased IFNβ-stimulated pSTAT1 association with STAT2 and IRF9 in the presence of vIRF-1 indicates that vIRF-1 may suppress pSTAT1 via release of the transcription factor from ISGF3 or STAT1-STAT2 heterodimers, thereby promoting dephosphorylation of pSTAT1. Detection of intracellular complexes containing vIRF-1, pSTAT2 and IRF9 and dissociation of IFNβ-induced STAT1-STAT2 interaction by vIRF-1 in vitro are consistent with this model. Data from in vitro competition and STAT1/IRF9 and IRF9/STAT2 serial precipitation experiments provide evidence that vIRF-1 may also destabilizes IRF9-STAT2 association and dissociate ISGF3 interactions. It is possible that vIRF-1 also inhibits ISGF3 complex formation (not illustrated), via (detected) interactions with pre- and/or post-activated STAT1, STAT2 and IRF9, but the presented data do not specifically address this. In the diagram, blue lines indicate activities and consequences of vIRF-1.

Article Snippet: For immunoprecipitations, IRF9 antibodies from BioLegend (catalog number 660702) and Santa Cruz Biotechnologies (sc-365893) and STAT1 and STAT2 antibodies from Santa Cruz Biotechnologies (sc-464 and sc-1668) were used.

Techniques: Activation Assay, Phospho-proteomics, De-Phosphorylation Assay, Knock-Out, In Vitro

Journal: PLoS ONE

Article Title: Protective Roles of Interferon-Induced Protein with Tetratricopeptide Repeats 3 (IFIT3) in Dengue Virus Infection of Human Lung Epithelial Cells

doi: 10.1371/journal.pone.0079518

Figure Lengend Snippet: A549 cells were infected by mock or DV for 3, 6, and 24 h and protein levels of both phosphorylated and non-phosphorylated STAT1, STAT2, and STAT3 were analyzed by western blotting (A). Treatment with 1000 units IFN-α was used as a positive control. Expression of IFIT3 in DV-infected A549 cells with knockdown of either STAT2 (B), STAT1 (C) or STAT3 (D) was determined by western blotting (B, C, and D) or quantitative RT/PCR (B). Both shRNA and siRNA were used as the approaches for STAT1/STAT2 and STAT3, respectively, as described in Materials and Methods. Knockdown with shGFP or si-Ctl was used as a negative control. Data show representative results and analysis pooled from at least 3 independent experiments. The analysis was performed by ANOVA as described in Materials and Methods. **P < 0.01. Ctl stands for control.

Article Snippet: A variety of antibodies recognizing different molecules were purchased: IFIT3 and BAX (GeneTex Inc, Irvine, CA, USA); STAT1, STAT2, and STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phosphorylated STAT1, phosphorylated STAT2, phosphorylated STAT3, caspase 3, caspase 8, and caspase 9 (Cell Signaling, Beverly, MA, USA).

Techniques: Infection, Western Blot, Positive Control, Expressing, Knockdown, Quantitative RT-PCR, shRNA, Negative Control, Control