Journal: Immunity

Article Title: Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling.

doi: 10.1016/j.immuni.2005.01.005

Figure Lengend Snippet: Figure 4. MV-Induced STAT2 Activation Leads to Inhibition of DC Differentiation

Article Snippet: Western Blot Analysis Immunoblot analysis was performed with antibodies against STAT2, phospho-STAT2 (Tyr689) (Upstate), and Actin (Santa Cruz Biotechnology) as primary antibodies and a horseradish peroxidase-linked anti-rabbit or mouse immunoglobin G as a secondary antibody.

Techniques: Activation Assay, Inhibition

Journal: Immunity

Article Title: Viruses evade the immune system through type I interferon-mediated STAT2-dependent, but STAT1-independent, signaling.

doi: 10.1016/j.immuni.2005.01.005

Figure Lengend Snippet: Figure 5. LCMV Cl 13 Inhibits DC Development via STAT2 Signaling In Vivo

Article Snippet: Western Blot Analysis Immunoblot analysis was performed with antibodies against STAT2, phospho-STAT2 (Tyr689) (Upstate), and Actin (Santa Cruz Biotechnology) as primary antibodies and a horseradish peroxidase-linked anti-rabbit or mouse immunoglobin G as a secondary antibody.

Techniques: In Vivo

Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: Rabbit anti-phospho-Tyr-689-STAT2 Clone D3P2P , Cell Signaling Technology , Cat No. 88410; RRID: AB_2800123.

Techniques: Recombinant, Virus, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Extraction, Mutagenesis, Isolation, Reverse Transcription, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Conditions of basal ISG expression. a , b Bone marrow-derived macrophages (BMDM) isolated from wild-type (WT), Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were treated with 250 IU/ml of IFN-β as indicated. Gapdh-normalized gene expression was measured by RT-q-PCR. Data represent the mean and standard error of the mean (SEM) values of three independent experiments. P -values were calculated using the paired ratio t -test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). c Gene set enrichment analyses showing upregulation of an IFN and inflammatory response signature of untreated WT compared with untreated Irf9 −/− BMDM. The top correlated genes for each biological triplicate are displayed in the corresponding heat maps. The total height of the curve indicates the extent of enrichment (ES), with the normalized enrichment score (NES), the false discovery rate (FDR), and the P -value. Source data are provided as a source data file

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Expressing, Derivative Assay, Isolation, Gene Expression

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: IFN-induced gene expression and STAT complexes in BMDM. a – c panels on the left. Scatterplot linking RNA-seq ( n = 3) with ChIP-seq ( n = 2) data (BMDM). Differentially expressed genes (log-fold change (lfc) > 1, padj < 0.05) between Irf9 −/− and WT untreated ( a ), WT untreated versus WT IFN-β ( b ), or WT untreated versus IFN-γ-treated ( c ) BMDM are shown. Genes associated with complexes containing at least one of the ISGF3 subunits (STAT1, STAT2, and IRF9) according to ChIP with the respective antibodies are color-coded as follows. Blue triangles: STAT2–IRF9; red diamonds: STAT1, STAT2, and IRF9 (ISGF3); beige squares: STAT1 only. The pie chart inserts show the relative proportions of genes associated with STAT2–IRF9, ISGF3, or STAT1 dimers. Panels on the right d – f . Representative browser tracks of the ChIP-seq experiments shown in a – c ). Data from untreated ( d ), IFN-β (90 min; e ), or IFN-γ- (90 min; f ) treated BMDM derived from wild-type (WT) and Irf9 −/− ( IRF9 −/− ) mice are shown (scale 0–150). Individual tracks represent binding of STAT1 (S1/blue), STAT2 (S2/red), and IRF9 (green). Tracks are shown for ChIP-seq and control input, as well as regulatory chromatin sites from ATAC-seq for untreated BMDM, derived from data in ref.

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Gene Expression, RNA Sequencing, ChIP-sequencing, Derivative Assay, Binding Assay, Control

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: IFN-induced gene expression and STAT complexes in MEFs. a , b Log-fold change (lfc)/lfc plot comparing WT or IRF9 −/− BMDM with mouse embryonic fibroblast (MEF). mRNA expression ( n = 3) ratios, with a cutoff padj ≤ 0.05 and lfc ≥ 1, ( IRF9 −/− /WT) in resting ( a ) or in IFN-β- treated ( b ) cells is plotted. Genes affected by the loss of IRF9 in both cell types are displayed in blue. c , d GO analysis of the IRF9-dependent genes in untreated ( c ) and IFN-β-treated ( d ) cells as defined in a , b . The top seven pathways are listed for IRF9-dependent genes (in blue). e Representative genome browser tracks for transcription factor binding at ISG and control loci (scale 0–60): STAT1 (S1/blue), STAT2 (S2/red), and IRF9 (green). Tracks represent ChIP-seq experiments in untreated and IFN-β-treated MEF

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Gene Expression, Expressing, Binding Assay, Control, ChIP-sequencing

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Signal dependence of complex formation from ISGF3 subunits. a BMDMs from wild-type (WT) animals were treated for 1.5 h with IFN-β and immunoprecipitation was carried out using antibodies against STAT1, STAT2, IRF9, or an IgG control. Immunoprecipitated complexes were analyzed by western blotting with antibodies to STAT1, STAT2, IRF9, and GAPDH. Input controls represent 10% of the total lysate used for the immunoprecipitation. b The representative blot was quantified using ImageJ software. Relative intensities of the bands were normalized to their corresponding input sample. Data represent relative intensities in percent, where STAT1, STAT2, and IRF9 levels in IFN-β-treated IPs equal 100%. c Targeted quantitative MS analysis of STAT1, STAT2, and IRF9 BioIDs using PRM. Raw 264.7 cells were treated with 0.2 µg/ml doxycycline for 24 h, followed by addition of 50 µM biotin for 18 h. Cells were treated for 2 h either with IFN-β or IFN-γ lysed and protein complexes were isolated by streptavidin affinity purification, followed by analysis with LC–MS. Mean log 2 -transformed protein ratios were calculated for three biological replicates of myc-STAT1-BirA*, myc-STAT2-BirA*, or myc-IRF9-BirA* cells normalized to their appropriate localization control. Standard deviation and t -test statistics were calculated for each of the target proteins. P -values (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Source data are provided as a source data file

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Immunoprecipitation, Control, Western Blot, Software, Isolation, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Standard Deviation

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Complex formation of ISGF3 subunits and proximity labeling of interactors. a Raw 264.7 cells were treated for 1.5 h with IFN-β. Cell lysates were incubated with a biotinylated Oas1a-ISRE oligo, a biotinylated Isg15-ISRE oligo, or plasmid DNA. DNA-bound protein complexes were isolated by streptavidin affinity purification, followed by western blot analysis. b STAT1, STAT2, and IRF9 interactome dynamics in response to interferon treatment. Hierarchical cluster analysis of proteins significantly enriched upon treatment with IFN-β or IFN-γ. Proteins were filtered, which were at least twofold enriched above background (myc-BirA* or BirA*-NLS controls) in at least one condition at an adjusted p -value of < 0.01, and which showed at least a twofold increase in intensity after interferon induction when compared with steady-state conditions. For this filtered set of proteins, we computed the mean log 2 LFQ protein ratio of the interferon-induced (2 and 18 h) and the steady-state condition and used these values to generate a hierarchical cluster analysis and heat maps in Perseus with default settings. Interactor names shown in red were found associated with more than one ISGF3 subunit across all experimental conditions, including interactors under resting conditions shown in Supplementary Data . Source data are provided as a source data file

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Labeling, Incubation, Plasmid Preparation, Isolation, Affinity Purification, Western Blot

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Localization of STAT complexes in BMDM. a IRF9 localization as determined by immunofluorescence. BMDMs of wild-type (WT) and Irf9 −/− ( IRF9 −/− ) mice were left untreated or stimulated with IFN-β for 30 min. The cells were fixed and stained with an anti-IRF9 antibody followed by Alexa Fluor ® 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (magenta). First Ab (−) indicates the control without the first antibody. The scale bars represent 10 µm. b – d Nuclear and cytoplasmic extracts from BMDM were prepared from controls or after a 30-min treatment with IFN-β or IFN-γ and analyzed by western blot. A 2:1 ratio of the nuclear-to-cytoplasmic fraction is shown. Where indicated, 15 µM P6 inhibitor or DMSO were added for 3 h prior to IFN treatment. Phosphorylation of STAT1 at Y701 and total STAT1 levels, as well as phosphorylation of STAT2 at Y689, total STAT2 and IRF9, and α-tubulin and lamin A/C levels were determined. e The nuclear fractions of the representative blots b – d were quantified using ImageJ software. Relative intensities of the bands were normalized to their corresponding lamin C levels. Data represent relative intensities in percent, where STAT1, STAT2, and IRF9 levels in IFN-β-treated nuclear extracts equal 100%. f Phosphorylation of STAT1 at Y701, STAT2 at Y689, total STAT1, total STAT2, IRF9, and α-tubulin was determined in whole-cell lysates of BMDM. Overall, 15 µM P6 inhibitor or DMSO were added for 3 h prior to IFN treatment. Source data are provided as a source data file

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Immunofluorescence, Staining, Control, Western Blot, Phospho-proteomics, Software

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Cross-regulation of ISGF3 subunits. a BMDMs isolated from WT, Stat1 −/− , Stat2 −/− , and Irf9 −/− mice were left untreated or treated with 15 µM P6 inhibitor for 3 h. Gapdh-normalized gene expression was measured by RT-q-PCR. Data represent relative expression in percent, where WT untreated equals 100%. Data represent the mean and standard error of the mean (SEM) values of three independent experiments. P -values were calculated using the paired ratio t -test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). b STAT1 (S1), STAT2 (S2), and IRF9 binding at the Stat1 , Stat2 , and Irf9 promoters (scale 0–150). The browser tracks represent data derived from the ChIP-seq experiments in BMDM described in the legend of Fig. . c Whole-cell extracts from wild-type, Stat1 −/− , Stat2 −/− , and Irf9 −/− BMDMs were tested by western blot for total STAT1, STAT2, and IRF9 levels. d Whole-cell extracts from wild-type, Stat1 −/− , Stat2 −/− , and Irf9 −/− mouse embryonic fibroblasts were analyzed by western blot for total STAT1, STAT2, and IRF9 levels. e Stat1 −/− mouse embryonic fibroblasts were stably transduced with a doxycycline-inducible STAT1-myc construct. Whole-cell extracts from wild-type, Stat1 −/− , and reconstituted Stat1 −/− MEFs were tested by western blot for total STAT1, STAT2, and IRF9 levels in the absence and presence of doxycycline. f The representative blot in e was quantified using ImageJ. Relative intensities of the bands were normalized to their corresponding GAPDH levels. Data represent relative intensities in percent, where STAT1, STAT2, and IRF9 levels in untreated wild-type MEFs equal 100%. Source data are provided as a source data file

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Isolation, Gene Expression, Expressing, Binding Assay, Derivative Assay, ChIP-sequencing, Western Blot, Stable Transfection, Transduction, Construct

Journal: Nature Communications

Article Title: A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription

doi: 10.1038/s41467-019-10970-y

Figure Lengend Snippet: Model of the molecular switch from resting to IFN-induced gene expression. Under homeostatic conditions, a tonic signal from the type I-IFN receptor activates small quantities of ISGF3, which increase basal expression of the genes encoding the ISGF3 subunits STAT1, STAT2, and IRF9. This causes the constitutive formation of STAT1–STAT2 as well as STAT2–IRF9 complexes. Basal expression of a large fraction of interferon-induced genes (ISGs) is stimulated by STAT2–IRF9 complexes that appear in the nucleus without a signaling requirement. Signaling by the type I-IFN receptor and to a significant extent also by the IFN-γ receptor causes the formation of tyrosine-phosphorylated STAT1–STAT2 heterodimers that translocate to the nucleus and form an ISGF3 complex by associating on DNA with IRF9. The higher off-rate of STAT2–IRF9 compared with ISGF3 combined with a larger quantity of tyrosine-phosphorylated STAT1–STAT2 versus STAT2–RF9 complexes in IFN-treated cells most likely explains why a rapid exchange takes place after interferon treatment

Article Snippet: Two antibodies against Stat1 were used in experiments with BMDM and MEFs, respectively (Cell Signaling, Catalog # 9172 1:1000 and Santa Cruz, Catalog # sc-346; 1:1000); Stat2 (Cell Signaling, Catalog # 72604, 1:1000); α-Tubulin (Sigma, Catalog # T9026, 1:5000); Phospho-Stat1 (Tyr701; Cell Signaling, Catalog # 9167, 1:1000); Phospho-STAT2 (Tyr689, Merck, Catalog # 07-224, 1:1000); Lamin A/C (Santa Cruz, Catalog # sc-376248, 1:1000); GAPDH (Millipore, Catalog # ABS16, 1:3000); IRF9 (6F1, hybridoma supernatants used for experiments with mouse cells, 1:20); IRF9 (used for experiments with THP-1 cells, Santa Cruz, Catalog # sc-10793, 1:1000); anti-myc (Cell Signaling, Catalog # 2276, 1:1000).

Techniques: Gene Expression, Expressing

Journal: Viruses

Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

doi: 10.3390/v10120726

Figure Lengend Snippet: Expression of MetYPCP of HEV ORF1 inhibited STAT1 but not STAT2 phosphorylation upon IFN-β treatment. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 500 IU/mL of IFN-β. Cell lysates were extracted and used for the detection of FLAG-tagged proteins, total STAT1, phosphorylated STAT1 (p-STAT1), total STAT2 and phosphorylated STAT2 (p-STAT2) by immunoblotting. Actin served as an internal control. ( b ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP, PCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT1, p-STAT1 and actin by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT1, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT1 and actin, STAT1 and actin, and p-STAT1 and STAT1 were calculated and expressed as a relative percentage in comparison to the EV control. ( c ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT2, p-STAT2 and p-STAT1 by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT2, p-STAT2, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT2 and actin and STAT2 and actin were calculated and expressed as a relative percentage in comparison to the EV control. The ratio between p-STAT1 and actin was also determined to ensure significant inhibition of the p-STAT1 level by MetYPCP in this set of experiments. In ( b – c ), the mean percentage (± standard deviation) of four independent experiments is presented for each panel: * p < 0.05; ** p < 0.005; *** p < 0.0005 compared to the EV control for IFN-treated samples (unequal variance t -tests).

Article Snippet: Polyclonal antibodies against STAT1 (06-501), phospho-STAT1 (Tyr701) (07-307) and phospho-STAT2 (Tyr 689) (07-224) were from Merck Millipore (Darmstadt, Germany).

Techniques: Expressing, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Control, Software, Comparison, Inhibition, Standard Deviation

Journal: The Journal of general virology

Article Title: Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation.

doi: 10.1099/vir.0.2008/001669-0

Figure Lengend Snippet: Fig. 1. (a) MRC-5 cells were infected with HCMV strains AD169 and Towne (5 p.f.u. per cell) and cell lysates were prepared at the indicated time points p.i. Equivalent amounts of lysate were separated by SDS-PAGE and immunoblotted using antibodies against STAT2 and STAT1. HCMV pp65 and pp72, as infection controls, and b-actin, as a loading control, were detected by reblot of the same membrane. (b) MRC-5 cells were infected with HCMV strain TB40/E and the clinical isolates UL1271 and UL1702 (5 p.f.u. per cell). Cell lysates were prepared at the indicated time points p.i. Equivalent amounts of lysate were analysed as described above.

Article Snippet: Immunoblot analyses were performed with the following antibodies: monoclonal anti-b-actin antibody produced in mouse (Sigma); mouse monoclonal antibody (mAb) against HCMV pp65 (3A12; AbCam); rabbit antiserum specific for STAT1 (E23; Santa Cruz), STAT2 (06-502; UpstateBiotechnology), STAT3 (C20; Santa Cruz), Jak1 (HR-785; Santa Cruz) or IRF-9/p48 (H-143; Santa Cruz); and mAb recognizing p-Tyr701 STAT1 (A2; Santa Cruz), p-Tyr689 STAT2 (Tyr 689; Upstate) or p-Tyr STAT3 (B7; Santa Cruz).

Techniques: Infection, SDS Page, Control, Membrane

Journal: The Journal of general virology

Article Title: Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation.

doi: 10.1099/vir.0.2008/001669-0

Figure Lengend Snippet: Fig. 2. (a) HCMV upregulates STAT2 mRNA synthesis. MRC-5 cells were infected with HCMV strain AD169 (5 p.f.u. per cell) and total RNA was prepared at the indicated time points. Equivalent amounts of RNA were subjected to agarose gel electrophoresis (lower panel) and subsequently analysed by Northern blot using a STAT2- or HCMV-IE2-specific probe (upper and middle panels, respectively). (b) MRC-5 cells were either mock-infected or infected with HCMV strain AD169 (3 p.f.u. per cell). At 24 h p.i., either DMSO or 10 mM lactacystin (LC) in DMSO was added. Cells were treated with the indicated IFN (500 U ml”1) for 30 min before lysis in EMSA buffer at 48 h p.i. Equivalent amounts of lysate were subjected to SDS-PAGE and immunoblotted using antibodies against phospho-STAT2 (pSTAT2) and STAT2 (detected from the same membrane). HCMV pp65 served as an infection control and b-actin as a loading control. (c) MRC-5 cells were either mock-infected or infected with HCMV strain AD169 (3 p.f.u. per cell). At 14 h p.i., 10 mM MG132 was added as indicated to one set of infected samples and CHX was added to all samples. Cells were lysed at the indicated time points after CHX addition. Equivalent amounts of lysate were subjected to SDS-PAGE and immunoblotted using antibodies against STAT2. (d) MRC-5 cells were either mock-infected or infected with rVV (3 p.f.u. per cell) expressing either M27 or UL27. Wild-type VV served as a control. Equivalent amounts of lysate were subjected to SDS-PAGE and immunoblotted using antibodies specific for STAT2, UL27-V5, M27-FLAG and b- actin. (e) MRC-5 cells were either mock-infected or infected with the BAC-derived HCMV strain TB40/E or the TB40/E-DUL27 deletion mutant (3 p.f.u. per cell). Cells were lysed 48 h p.i. Equivalent amounts of lysate were subjected to SDS-PAGE and immunoblotted using antibodies against STAT2 and STAT1. HCMV pp65 served as an infection control and b-actin as a loading control.

Article Snippet: Immunoblot analyses were performed with the following antibodies: monoclonal anti-b-actin antibody produced in mouse (Sigma); mouse monoclonal antibody (mAb) against HCMV pp65 (3A12; AbCam); rabbit antiserum specific for STAT1 (E23; Santa Cruz), STAT2 (06-502; UpstateBiotechnology), STAT3 (C20; Santa Cruz), Jak1 (HR-785; Santa Cruz) or IRF-9/p48 (H-143; Santa Cruz); and mAb recognizing p-Tyr701 STAT1 (A2; Santa Cruz), p-Tyr689 STAT2 (Tyr 689; Upstate) or p-Tyr STAT3 (B7; Santa Cruz).

Techniques: Infection, Agarose Gel Electrophoresis, Northern Blot, Lysis, SDS Page, Membrane, Control, Expressing, Derivative Assay, Mutagenesis

Journal: The Journal of general virology

Article Title: Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation.

doi: 10.1099/vir.0.2008/001669-0

Figure Lengend Snippet: Fig. 3. (a) STAT2 degradation is independent of HCMV particle components. MRC-5 cells were either mock-infected or infected with HCMV strain TB40/E (3 p.f.u. per cell) and compared with cells infected with UV-inactiv- ated TB40/E (5 p.f.u. per cell). Equivalent amounts of lysate were separated by SDS- PAGE and immunoblotted using antibodies against STAT2. (b) STAT2 degradation is an HCMV early gene function. MRC-5 cells were either mock-infected or infected with HCMV strain AD169 (3 p.f.u. per cell). PAA (250 mg ml”1) was added at the time point of infection until 72 h p.i. Cell lysates were prepared at the indicated time points p.i., equivalent amounts were separated by SDS-PAGE and immuno- blotted using antibodies against STAT2. (c) MRC-5 cells were either mock-infected or infected with HCMV strain TB40/E (3 p.f.u. per cell). If indicated, 50 mg CHX ml”1 was added at the same time as infection. At 3 h p.i., CHX was washed out and replaced by 5 mg actinomycin D ml”1 which was left on cells for the duration of the experiment. Cell lysates were prepared at the indicated time points p.i. Equivalent amounts of lysate were separated by SDS-PAGE and immunoblotted using antibodies against STAT2. In (a–c), IE1/ pp72, as an infection control, and b-actin, as a loading control, were detected from the same membrane.

Article Snippet: Immunoblot analyses were performed with the following antibodies: monoclonal anti-b-actin antibody produced in mouse (Sigma); mouse monoclonal antibody (mAb) against HCMV pp65 (3A12; AbCam); rabbit antiserum specific for STAT1 (E23; Santa Cruz), STAT2 (06-502; UpstateBiotechnology), STAT3 (C20; Santa Cruz), Jak1 (HR-785; Santa Cruz) or IRF-9/p48 (H-143; Santa Cruz); and mAb recognizing p-Tyr701 STAT1 (A2; Santa Cruz), p-Tyr689 STAT2 (Tyr 689; Upstate) or p-Tyr STAT3 (B7; Santa Cruz).

Techniques: Infection, SDS Page, Control, Membrane

Journal: The Journal of general virology

Article Title: Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation.

doi: 10.1099/vir.0.2008/001669-0

Figure Lengend Snippet: Fig. 5. (a) MRC-5 cells were either mock-infected or infected with HCMV strain Towne (3 p.f.u. per cell). Cells were lysed after a 30 min period of induction with 500 U ml”1 of the respective IFN (indicated by +). Equivalent amounts of lysates collected at the indicated time points were subjected to SDS-PAGE and immunoblotted using antibodies against phospho-STAT2 (p- STAT2), STAT2, phospho-STAT1 (p-STAT1), STAT1, Jak1, phospho-STAT3 (p-STAT3), STAT3 and IRF-9/p48. STAT proteins were probed as a reblot from the same membrane used for the respective phospho-STAT proteins. HCMV pp65 and IE1/pp72 were used as infection controls and b-actin was used as a loading control. (b) MRC-5 cells were either mock-infected or infected with HCMV strain AD169 (3 p.f.u. per cell). STAT1 activation was monitored at the indicated time points by Western blot after a 30 min period of induction with 250 U IFN-c ml”1. Antibodies against phospho-STAT1, STAT1, IE1/pp72 and b- actin were used as described for (a). (c) MRC-5 cells were either mock-infected or infected with HCMV strain AD169 (3 p.f.u. per cell). Cells were treated for 30 min with 500 U IFN-a ml”1 before harvesting at the indicated time points. Equivalent amounts of lysates were subjected to SDS-PAGE and then immunoblotted using antibodies against phospho-STAT2, STAT2, IE1/pp72 and b-actin as described for (a).

Article Snippet: Immunoblot analyses were performed with the following antibodies: monoclonal anti-b-actin antibody produced in mouse (Sigma); mouse monoclonal antibody (mAb) against HCMV pp65 (3A12; AbCam); rabbit antiserum specific for STAT1 (E23; Santa Cruz), STAT2 (06-502; UpstateBiotechnology), STAT3 (C20; Santa Cruz), Jak1 (HR-785; Santa Cruz) or IRF-9/p48 (H-143; Santa Cruz); and mAb recognizing p-Tyr701 STAT1 (A2; Santa Cruz), p-Tyr689 STAT2 (Tyr 689; Upstate) or p-Tyr STAT3 (B7; Santa Cruz).

Techniques: Infection, SDS Page, Membrane, Control, Activation Assay, Western Blot