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: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: A , B Cytoplasmic retention of Flag-tagged WT STAT1 in IFNγ-stimulated cells co-expressing R602L/Y701F-GFP. Transfected STAT1-negative U3A and HeLa cells expressing R602L/Y701F-GFP and WT-Flag were treated with 50 ng/ml IFNγ for 45 min and subsequently stained with an anti-Flag antibody. A The fluorescence micrographs show the intracellular distribution of Flag-tagged STAT1 co-expressed in cells transfected with R602L/Y701F-GFP as well as the localization of the corresponding Hoechst-stained nuclei (n = 3 independent transfections). B The histogram demonstrates the nucleocytoplasmic STAT1-Flag distribution in the absence and presence of R602L/Y701F-GFP in IFNγ-treated HeLa cells, as determined by the ratio of nuclear-to-total fluorescence intensity (means ± standard deviations from n = 20 cells, * p ≤ 0.05, as assessed by a two-tailed Student’s t test). C The absence of nuclear P-STAT1 staining of the endogenous protein in HeLa cells expressing R602L/Y701F-GFP. HeLa cells were transfected with R602L/Y701F-GFP and stimulated with IFNγ for indicated times before staining with an anti-phospho-tyrosine antibody. D Crystal structure of the STAT1 anti-parallel dimer. A critical arginine residue at position 274 marked in cyan in the coiled-coil domains and threonine residue at position 385 marked in pink in the DNA-binding domains of the two STAT1 proteins (white and orange). Structural data were obtained from the Protein Data Bank (pdb) file 1YVL for the STAT1 anti-parallel dimer. E The absence of nuclear P-STAT1 staining of endogenous STAT1 in HeLa cells expressing R274W/R602L/Y701F-GFP. HeLa cells were transfected with R274W/R602L/Y701F-GFP and stimulated with IFNγ for indicated times before being stained with an anti-phospho-tyrosine antibody. Scale bars in A , C , E mark a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Expressing, Transfection, Staining, Fluorescence, Two Tailed Test, Residue, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: The dimerization-deficient quadruple mutant (QM-GFP) mimicking U-STAT1 inhibits the detection of co-expressed WT protein but does not affect tyrosine phosphorylation. A – C HeLa cells were transfected with a plasmid coding for QM-GFP and stimulated with IFNγ for 45 min followed by staining with anti-phospho-tyrosine antibody. A The fluorescence micrographs show the intracellular distribution of QM-GFP and WT P-STAT1, as well as the localization of the corresponding Hoechst-stained nuclei. B Histogram demonstrating the net reduction of nuclear P-STAT1 intensity in HeLa cells expressing QM-GFP, as determined by the ratio of nuclear-to-total fluorescence intensity (n = 3 independent transfections, means ± standard deviations from n = 20 cells, * p ≤ 0.05 by a two-tailed Student’s t test). C Concentration-dependent inhibition of nuclear P-STAT1 accumulation by co-expressed QM-GFP. The fluorescence intensities of nuclear P-STAT1 staining were plotted against the total cellular QM-GFP fluorescence. D , E Representative immunoblot of whole cell extracts from STAT1-negative U3A cells co-expressing untagged STAT1 and QM-GFP after treatment for 45 min with 50 ng/ml of recombinant IFNγ and the quantification thereof from three independent transfection experiments. F , G An in vitro phosphorylation assay demonstrated no difference in tyrosine phosphorylation rates of the WT STAT1 by JAK2 with respect to the presence or absence of QM-GFP. Whole cell extracts (10 μl in each reaction) from reconstituted U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP were incubated with 4 μg/ml of recombinant JAK2 kinase and the levels of P-STAT1 were monitored over time by means of Western blotting (n = 3). Statistical analysis revealed no significant difference in the phosphorylation kinetics of the WT in the presence of either QM-GFP or WT-GFP. H , I Results from an in vitro dephosphorylation assay using extracts from IFNγ-pre-stimulated U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP (10 μl each) incubated for 0, 15 and 30 min with 2 U of the STAT-specific Tc45 phosphatase (n = 3). Tyrosine dephosphorylation was followed by immunoblotting including a quantitative analysis of the phospho-tyrosine signals divided by total amount of STAT1 signal. Scale bar in A marks a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Staining, Fluorescence, Expressing, Two Tailed Test, Concentration Assay, Inhibition, Western Blot, Recombinant, In Vitro, Incubation, De-Phosphorylation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Unaltered DNA binding, reporter activation and target gene induction by WT STAT1 in the presence of a dimerization-deficient mutant. A Electrophoretic mobility shift assay demonstrated unchanged binding of the recombinant WT protein from reconstituted U3A cell extracts to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, whereas the co-expressed unphosphorylated STAT1 variants and empty vector (pEGFPN1) showed no DNA-binding indicated by the absence of a GFP-tagged STAT1 band in lanes 3 to 5. The asterisk at the margin of the gelshift indicates an unspecific band. B EMSA result showing the unaltered capacity of endogenous STAT1 from HeLa cell extracts to form tetramers on the radioactively-labelled DNA probe 2xGAS, containing two GAS sites in tandem orientation, in the presence of co-expressed QM-GFP. HeLa cells were either untransfected (UT) or transfected with the indicated expression plasmids, and on the next day the cells were treated for 45 min with IFNγ or left untreated. Extracts were incubated with 2xGAS DNA probed for 15 min before subsequently being challenged by a 750-fold molar excess of unlabelled M67 DNA incubated for 10 min at RT in a competition assay (comp). For super-shift assays, 20 ng of STAT1- (αS1) or STAT3-specific (αS3) antibody were added to the reaction for 40 min at RT. C , D EMSA result showing the unaltered cytoplasmic and nuclear fractions of endogenous P-STAT1 from HeLa cell lysates bound to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, as the additional presence of QM-GFP did not hamper nuclear import of the endogenous STAT1 but its nuclear retention. E HeLa cells expressing WT-GFP, empty vector (pEGFPN1) or the quadruple mutant QM-GFP were left untreated (−) or stimulated for 6 h with 50 ng/ml of IFNγ (+). In whole extracts from these cells, luciferase luminescence of a reporter construct with a triple GAS site (3xLy6E) and the enzymatic activity of the co-expressed β-galactosidase were measured and represented graphically. F U3A cells were transfected with a STAT1-responsive luciferase reporter construct, a β-galactosidase expression vector and a combination of equal amounts of WT-GFP and the indicated STAT1 variants. These cells were either unstimulated (−) or stimulated with 50 ng/ml of IFNγ (+) and subsequently luciferase activity, normalized to the β-galactosidase expression, was measured in whole cell extracts and represented graphically. The experiment was repeated in six independent transfections at least three times. G – J U3A cells were untransfected (UT) or transfected with a plasmid coding for WT-GFP alone or a combination of WT-GFP and the indicated U-STAT1 variants or empty GFP vector (pEGFPN1). These cells where either untreated or stimulated with 50 ng/ml of IFNγ for 6 h. after which RNA was isolated and converted to cDNA. Histograms show the results from qPCR experiments for the following STAT1 target genes: G GBP1 , H MIG , I CXCL10 and J IRF1 . Gene induction was normalized to the expression of the house-keeping gene GAPDH . Histograms show means and standard deviations wherein the significant differences between the IFNγ-stimulated variant samples and cells expressing a single transfection of WT protein are marked by bars and asterisks. The experiment was repeated three times

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Binding Assay, Activation Assay, Mutagenesis, Electrophoretic Mobility Shift Assay, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, Incubation, Competitive Binding Assay, Luciferase, Construct, Activity Assay, Isolation, Variant Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Deletion of the amino-terminus in mutant U-STAT1 restores nuclear accumulation of co-expressed, endogenous P-STAT1. A , B HeLa cells were transfected with an expression plasmid coding for R602L/Y701F-GFP, QM-GFP, ΔN/R602L/Y701F-GFP and QM/L407A/L409A-GFP and stimulated for 0 min or 45 min with 50 ng/ml of recombinant IFNγ, followed by staining with an anti-phospho-tyrosine antibody. Fluorescence micrographs show restored nuclear accumulation of P-STAT1 in the presence of amino-terminal deletion mutant ΔN/R602L/Y701F-GFP and the dsNLS mutant QM/L407A/L409A-GFP. Histograms show the quantification of nuclear P-STAT1 from HeLa cells co-expressing the indicated STAT1 variants in comparison with untransfected HeLa cells from three independent experiments (n = 3, means ± standard deviations from n = 20 cells, * p ≤ 0.05). C Fluorescence micrographs displaying the absence of nuclear endogenous P-STAT1 staining in the presence of mutant U-STAT1 constructs expressed in HeLa cells stimulated for 0 min and 45 min with recombinant IFNα (50 ng/ml). Note the rescue effect mediated by the deletion of the amino-terminus in the construct ΔN/R602L/Y701F-GFP under stimulation with type I IFN. Scale bars in A , C mark a distance of 10 µm. D Cellular fractionation experiments using cytoplasmic and nuclear lysates from HeLa cells expressing the indicated STAT1 variants demonstrate the presence of endogenous phospho-STAT1 in both compartments in IFNγ-pretreated cells. Representative immunoblots demonstrate unchanged tyrosine phosphorylation of endogenous STAT3 in whole cell extracts from HeLa cells expressing U-STAT1, after stimulation with IFNγ (lower panels). E , F Quantification from immunoblots of whole extracts from HeLa cells expressing the indicated mutants, including the quadruple mutant (QM), before and after treatment for 45 min with 50 ng/ml of recombinant IFNγ from three independent transfection experiments for the expression of phospho-STAT1 ( E ) and the expression of phospho-STAT3 ( F )

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Recombinant, Staining, Fluorescence, Comparison, Construct, Cell Fractionation, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Proposed model of U-STAT1-dependent import modulation of P-STAT1. The left panel describes a condition of low-level cellular U-STAT1, wherein fully functional import complexes of P-STAT1 are formed and translocated to the nucleus in cytokine-stimulated cells. The right panel depicts a high concentration of U-STAT1, wherein U-STAT1 interacts through its amino-terminus and dsNLS with both components of the importin dimer. The formation for a full-fledged import complex is hindered by the missing tyrosine phosphorylation residue of U-STAT1

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Functional Assay, Concentration Assay, Phospho-proteomics, Residue

Journal: PLoS ONE

Article Title: SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing

doi: 10.1371/journal.pone.0212053

Figure Lengend Snippet: A) Schematic overview of experimental design in B. B) Growth kinetics of cells left untreated or treated with 10 ng/ml of IFN-γ for 24 h, in the presence or absence of Q-VD-OPh. C). Western blot analysis of phosphorylated STAT1 in HAP1 cells transduced with a control lentiviral vector or with lentiviral vectors encoding independent SLFN11-targeting shRNA, either left untreated or exposed to 10 ng/ml of IFN-γ for 24h. D) IRF1 transcript levels following exposure to 10 ng/ml of IFN-γ for the indicated times in parental, IFNGR1 KO, and SLFN11 KO cells.

Article Snippet: The following antibodies were used for western blot analysis: anti-HSP90: H114 (SantaCruz); anti-TUBA1A: 2144s (Cell Signaling Technology); anti-phosphorylated STAT1 7649s (Cell Signaling Technology); anti γ-H2AX 2577s (Cell Signaling Technology); anti-phosphorylated CHK1 12302s (Cell Signaling Technology); anti-phosphorylated ATM 10H11.E12 (Millipore); anti-SLFN11 HPA023030 (Atlas).

Techniques: Western Blot, Transduction, Control, Plasmid Preparation, shRNA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide.

doi: 10.4049/jimmunol.165.6.3051

Figure Lengend Snippet: FIGURE 4. VIP and PACAP inhibit the IFN-g-induced tyrosine phos- phorylation of STAT1. Raw 264.7 cells (3 3 107 cells) were incubated with medium alone (unstimulated) or activated with 100 U/ml IFN-g in the presence or absence of 1028 M VIP or PACAP. At different times, cell extracts (30 mg protein) were immunoprecipitated (IP) with anti-STAT1 Ab and separated by 10% SDS-PAGE. Proteins were transferred to mem- branes, and the blots were developed with an Ab specific to phosphorylated STAT1 (upper panel). To determine the levels of STAT1 immunoprecipi- tated in each case, the membranes were stripped and reprobed with Abs directed to STAT1 (lower panel). Similar results were obtained in three separate experiments.

Article Snippet: Abs against IRF-1, phospho-Tyr (PY20), phosphorylated-STAT1 (Tyr701), Jak1, Jak2, STAT1a p91, and NF-kB p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Immunoprecipitation, SDS Page

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide.

doi: 10.4049/jimmunol.165.6.3051

Figure Lengend Snippet: FIGURE 3. VIP and PACAP decrease STAT1 binding to the IRF-1 promoter. A, Raw 264.7 cells (107 cells) were incubated with medium alone (un- stimulated) or activated with 100 U/ml IFN-g in the presence or absence of 1028 M VIP or PACAP. After 45 min incubation, nuclear extracts were pre- pared and analyzed for DNA binding activity by EMSA using radiolabeled oligonucleotides con- taining the GAS sequence motif of the IRF-1 pro- moter. Specificity was assessed by the addition of 50-fold excess of unlabeled homologous (GAS), or nonhomologous (NF-kB) oligonucleotides (Comp). B, Identification of the proteins bound to the GAS site. Nuclear extracts were incubated with poly- clonal Abs against STAT1 or NF-kB p65 for 20 min before the addition of the oligonucleotide probe. The arrow indicates the supershifted STAT1 band. Similar results were observed in three inde- pendent experiments.

Article Snippet: Abs against IRF-1, phospho-Tyr (PY20), phosphorylated-STAT1 (Tyr701), Jak1, Jak2, STAT1a p91, and NF-kB p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Incubation, Activity Assay, Sequencing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide.

doi: 10.4049/jimmunol.165.6.3051

Figure Lengend Snippet: FIGURE 6. Involvement of VPAC1 and cAMP signaling in the inhib- itory effects of VIP on Jak1-STAT1 activation. Raw 264.7 cells (3 3 107

Article Snippet: Abs against IRF-1, phospho-Tyr (PY20), phosphorylated-STAT1 (Tyr701), Jak1, Jak2, STAT1a p91, and NF-kB p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Activation Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 1. Effects of ethanol metabolism on STAT1 phosphorylation in VL-17A cells. VL-17A cells treated or not with 100 mmol/L ethanol in the presence or absence of 5 mmol/L 4MP, 10 mol/L DAS, or 100 mol/L UA for 72 hours and then exposed to IFN for 1 hour before harvest. Cells were lysed, immunoblotted, and probed for pSTAT1 and STAT1, as described in Methods. The results are presented as percent of control (IFN alone) of pSTAT1/STAT1 ratio. The data represent the mean SD from four independent experiments. * indicates a significant difference (P .05) between IFN-treated samples and other treatments. Inset: Representative blot of immunoreactive pSTAT1 and STAT1. STAT1, signal transducer and activator of transcription 1; DAS, diallyl sulfide; IFN, interferon gamma.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 3. Effects of BSO on total GSH levels and on STAT1 phosphory- lation in control and ethanol-treated VL-17A cells. (A) VL-17A cells were treated with 0, 0.1, or 1 mmol/L BSO for 48 hours. Data from 3 independent experiments with duplicate determinations are presented. *Significant difference (P .05) between control and BSO-treated samples. (B) VL-17A cells were exposed or not to either 100 mmol/L EtOH or 0.1 mmol/L BSO or to both agents for 48 hours and then treated with IFN. Data from 3 experiments are presented. *Significant (P .05) difference between IFN-treated samples compared with other samples in set. Inset: representative blot. BSO, L-buthionine sulfoximine; GSH, glutathione; STAT1, signal transducer and activator of transcription 1; IFN, interferon gamma.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 2. Time course of ethanol effects on IFN-induced STAT1 phos- phorylation (A), CYP2E1 activity (B), and comparison of total GSH levels in VL-17A cells, HepG2 cells, and hepatocytes (C). (A) Cells were treated or not with 100 mmol/L EtOH for 24, 48, and 72 hours and then exposed to IFN (IFN bar shows no exposure to ethanol, E 24 hIFN shows exposure to ethanol for 24 hours, E48hIFN is 48 hours’ ethanol exposure and 72hIFN is 72 hours’ ethanol exposure before treatment with IFN). pSTAT1 and STAT1 were determined as described in Methods. Data presented are from 3 independent experiments as mean SD. * indicated a significant difference (P .05) between IFN-treated sam- ples and IFNEtOH-treated samples. (B) Two sets of VL-17A cells were treated with 100 mmol/L EtOH for 0, 24, 48, and 72 hours followed by 1 hour exposure to IFN. CYP2E1 activity was determined in 1 set of cells. In the other set of cells, the pSTAT1/STAT1 ratio was measured in lysates at each time point. Data are presented as CYP2E1 specific activity and the percent control (IFN alone) of the pSTAT1/STAT1 densitometric ratio. (C) Cells (HepG2 and VL-17A cells) were plated at 1 10 6/well in 6-well plates for 24 to 48 hours, then harvested, lysed, and precipitated with 5% TCA (4 experiments with duplicate determina- tions). Hepatocytes were freshly isolated from 9 rats, each maintained on the Lieber-DeCarli control diet. Total glutathione was determined, as described in Methods. *Significant (P .05) difference between VL-17A cells and other cells. STAT1, signal transducer and activator of transcrip- tion 1; GSH, glutathione; IFN, interferon gamma; TCA, trichloracetic acid.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activity Assay, Comparison, Control, Isolation

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 4. Effects of ethanol on STAT1 phosphorylation in VA-13 cells and in E-47 cells. (A) VA-13 cells were treated or not with 100 mmol/L ethanol for 72 hours and then exposed to IFN for 1 hour. pSTAT1/STAT1 ratio was determined by Western blot. Data from 3 experiments are presented as percentage of control (IFN-treated) pSTAT1/STAT1 ratio. *Significant (P .05) difference between IFN-treated samples and ethanol treatments. Inset: Representative blot. (B) E-47 cells with higher (17 5.2 units/mgP) or lower (7.3 2.5 units/mgP) CYP2E1 specific activity were treated or not with 100 mmol/L ethanol for 72 hours and then exposed to IFN for 1 hour. pSTAT1/STAT1 ratio was determined by Western blot. Data from 6 experiments (3 for cells with high CYP2E1 activity and 3 for cells with low CYP2E1 activity) are presented as the percentage of control of pSTAT1/STAT1 ratio. *Significant (P .05) difference between the indicated value and IFN-treated samples. Inset: representative blot. STAT1, signal transducer and activator of transcrip- tion 1; IFN, interferon gamma.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, Western Blot, Control, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 7. Effects of ethanol and SIN-1 on IFN-induced STAT1 phos- phorylation on Tyr and Ser residues in VL-17A cells. (A) VL-17A cells were treated or not with 100 mmol/L EtOH for 72 hours and then exposed to IFN for 1 hour before harvest. pSTAT1 Tyr, pSTAT1 Ser, and STAT1 were each determined by Western blot. Data from 3 independent experiments are presented as the mean StDev percent of control of pSTAT1/STAT1 ratio *Significant difference (P .05) between IFN-treated and IFNethanol-treated samples. Inset: Representative blot. (B) and (C) VL-17A cells were treated or not for 24 hours with 0.1 and 1 mmol/L SIN-1 and then exposed to IFN for 1 hour. STAT1 phosphorylation on Tyr 701 (A) and Ser 751 (B) was determined by Western blot. Data from 3 experiments are presented as percent of control of pSTAT1/STAT1. *Significant (P .05) difference between IFN treated and SIN-1IFN treated samples. Inset: Representative blot. IFN, interferon gamma; STAT1, signal transducer and activator of transcription 1.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Western Blot, Control, Phospho-proteomics

Journal: Hepatology (Baltimore, Md.)

Article Title: Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells.

doi: 10.1002/hep.20909

Figure Lengend Snippet: Fig. 8. The proposed effects of EtOH metabolism on IFN-induced STAT1 phosphorylation in VL-17A cells. Gray arrows show positive effects, black arrows show negative effects, X shows blockade. Ethanol (EtOH) is metabolized by ADH and CYP2E1 to acetaldehyde (Ach) and ROS/RNS, respectively. These reactive intermediates can damage mitochondria, thereby increasing superoxide production. Because ethanol also induces iNOS, causing nitric oxide (NO) formation, there is increased a formation of PN (ONOO), a reaction product of O2--induced STAT1 phosphory- lation. Stabilization of SOCS1 is one reason for impaired IFN signaling. In addition, PN reacts with STAT1 on Tyr residues, making them unavail- able for phosphorylation. IFN, interferon gamma; STAT1, signal trans- ducer, and activator of transcription 1. IFN, interferon gamma; STAT1, signal transducer and activator of transcription 1.

Article Snippet: Antibodies to phosphorylated STAT1 (one specific for Tyr 701 and the other for Ser 727) were from Cell Signaling (Beverly, MA); antibodies to the STAT1 protein were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics

Journal: PLoS Pathogens

Article Title: Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

doi: 10.1371/journal.ppat.1004924

Figure Lengend Snippet: A) A549 cells were transfected with PB2 ∆ or empty vector and subsequently infected with 5 MOI KAN-1. At the time points indicated total RNA was isolated and expressional changes of IFNβ or IP10 mRNAs were analyzed by qRT-PCR and are depicted as mean n -fold (±SD) of one representative out of two independent experiments normalized to empty vector-transfected control. Efficient transfection was confirmed by detection of PB2 ∆ mRNA. *p≤0.05; ***p≤0.001; two-way ANOVA, Sidak’s multiple comparisons test. B, F) A549 or C) Vero cells were transfected with PB2 ∆ or empty vector and subsequently infected with B) 0.01 MOI KAN-1, C) 0.001 MOI KAN-1 or F) 0.01 MOI VSV. At the time points indicated virus-containing supernatants were harvested and infectivity titers were determined by a standard plaque assay and are depicted as mean (±SD) of three independent experiments. **p≤0.01, ***p≤0.001; two-way ANOVA, Sidak’s multiple comparisons test. D, E) A549 cells were transfected with PB2 Δ or empty vector and 32 h p.t. conditioned media were transferred to IFNAR2 siRNA-transfected A549 cells or were enriched with neutralizing IFNβ antibody and transferred to untreated A549 cells. 16 h p.s. acceptor cells were infected with 0.1 MOI rKAN-1 WT for 24 h. D) Infectivity titers were determined by standard plaque assay and are depicted as mean (±SD) of one representative out of three independent experiments. *p≤0.05; **p≤0.01; ***p≤0.001; two-way ANOVA, Dunnett’s multiple comparisons test relating to empty vector-conditioned media-treated control cells, respectively. E) Expression of viral proteins PB2, NP, M1 and NS1 in conditioned medium-treated acceptor cells upon IFNAR2 knockdown or IFNβ neutralization was analyzed by Western blot. Knockdown and neutralization efficiencies were verified by using phosphor-STAT1 Tyr701 antibodies. Tubulin expression served as loading control. Blots are representative of three independent experiments.

Article Snippet: Antibody against phosphor-STAT1 Tyr701 (clone 14) was purchased from BD Transduction Laboratories.

Techniques: Transfection, Plasmid Preparation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay, Expressing, Neutralization, Western Blot

Journal: PLoS Pathogens

Article Title: Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

doi: 10.1371/journal.ppat.1004924

Figure Lengend Snippet:

Article Snippet: Antibody against phosphor-STAT1 Tyr701 (clone 14) was purchased from BD Transduction Laboratories.

Techniques: