Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 1. PIV5 V protein binds human but not mouse STAT2. Mouse NIH3T3 cells or human 2fTGH and HEK293Tcells were transfected with either FLAG-tagged PIV5 V protein (V) or FLAG vector control (C). Parallel samples of NIH3T3 were co-transfected with human STAT2 expression vector (hST2). Whole cell extracts were immunoprecipitated with FLAG-M2 agarose beads, eluted with SDS–PAGE buffer and processed for immunoblot with specific antibodies indicated.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Immunoprecipitation, SDS Page, Western Blot

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 2. Expression of human STAT2 in transgenic mice. (A) The 2555-bp open reading frame encoding human STAT2 was flanked by the human ubiquitin C promoter on the 5′ end and an SV40 splice site and poly A tail on the 3′ end. (B) NIH3T3 cells were transfected with the 5xISRE-luciferase plasmid with or without the hSTAT2 transgene construct, infected with PIV5 for 24 h prior to luciferase assay. Normalized to co-transfected Renilla luciferase. Bars indicate the mean normalized to percent of maximum (n=3) and error bars indicate standard deviation. (C) Ubiquitous STAT2 expression in transgenic mice. Protein extracts (20 μg) from isolated transgenic mice were subject to immunoblot to detect human STAT2. Ubiquitous expression of the transgene throughout the mouse was observed.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Expressing, Transgenic Assay, Ubiquitin Proteomics, Transfection, Luciferase, Plasmid Preparation, Construct, Infection, Standard Deviation, Isolation, Western Blot

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 3. Human STAT2 activity in mouse does not alter IFN signaling. (A) Splenocytes from transgenic and wild-type mice were treated with murine IFNβ for 5–30 min. Cells were immediately lysed and processed for immunoblot with antibodies that recognize human and mouse tyrosine phosphorylated STAT2, human STAT2, mouse STAT2 and STAT1α/β (recognizes human and mouse). (B) Splenocytes from transgenic and wild-type mice were treated with IFNβ for 10 min prior to lysate preparation and anti-STAT1 immunoprecipitation. Precipitated proteins were separated by SDS–PAGE and processed for immunoblot with antibodies for human STAT2, mouse STAT2 and tyrosine phosphorylated STAT2. (C) Splenocytes from hSTAT2 transgenic and wild-type mice were isolated and treated with mIFNβ for 6 or 18 h prior to RNA isolation and reverse transcription. Real-time PCR with primers specific for Mx1 and Ifi47 were performed and normalized to GAPDH. Graphs indicate average values for n=3, with error bars to represent standard deviation. (D) Transgenic and wild-type MEFs were pretreated 2 h with murine IFNβ, then infected with VSV (1 pfu/cell) for 16 h. Infectious virus released into the supernatant was estimated by titration on CV1 cells. IFN treatment provides a similar level of protection in transgenic and wild-type cells. Graph shows data from an individual VSV titration experiment. (E) Splenocytes were isolated from transgenic and wild-type mice and stimulated with IFNβ for 20′ prior to lysis. Whole cell extracts were separated by SDS–PAGE and immunoblotted with antibodies to detect STAT4 and tyrosine phosphorylated STAT4. IFNβ induces STAT4 activation in both wild-type and transgenic splenocytes.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Activity Assay, Transgenic Assay, Western Blot, Immunoprecipitation, SDS Page, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Infection, Virus, Titration, Lysis, Activation Assay

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 5. Transgenic mouse cells support enhanced PIV5 replication. (A) MEFs were infected with PIV5 for 24 h before additional (1000 U/ml) exogenous IFNβ for another 24 h. Cells were lysed and processed for immunoblot with human STAT2 and P/Vantibodies. (B) PIV5 titer from MEFs infected at low MOI (1 pfu/cell) with PIV5 after 24 and 48 h. Viral supernatant was titered by serial dilution on CV-1 cells. Results show greater viral replication in transgenic MEFs.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Transgenic Assay, Infection, Western Blot, Serial Dilution

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 2. IFN-inducible tyrosine phos- phorylation of STAT1 and intact and mutant STAT2 proteins. A, cells were either left untreated () or treated () for 15 min with 5 ng/ml IFN alfacon-1, as indicated. Protein lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-STAT2 antibody. Mem- branes were stripped and re-probed with anti-STAT2 and anti--actin antibodies to control for loading. B, similar to A, but antibodies were specific for phosphoryl- ated and total forms of STAT1.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Mutagenesis, SDS Page, Control

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 3. Cells expressing the VV-II STAT2 exhibit defective IFN-induced biological responses and GAS-mediated transcriptional activation. A, 104 cells were treated with the indicated doses of IFN alfacon-1 for 16 h, and then challenged with EMCV. 24 h later, virus-induced cytopathic effects were quantitated spectrophotometrically. Data are expressed as percent protection from the cytopathic effects of EMCV compared with untreated, uninfected controls. Mean value S.E. of three independent experiments is shown. B, 2 103 cells were treated with the indicated doses of IFN alfacon-1 for 72 h. Cell proliferation was assessed spectrophotometrically. Data are expressed as percent inhibition compared with untreated, control cells. Mean value S.E. of three independent experiments is shown. C, ISRE luciferase gene reporter constructs were co-transfected into cells with a construct carrying the -galactosidase gene. 48 h after transfection, cells were either left untreated or treated with 5 ng/ml IFN alfacon-1 for 6 h. Luciferase activity was measured and values were normalized for the -galactosidase activity of each sample to control for transfection efficiency. Data are expressed as -fold increase of luciferase activity in response to IFN over untreated samples. Values are mean S.E. of three independent experiments. D, similar to C, but cells were transfected with an 8GAS luciferase gene reporter construct.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activation Assay, Virus, Inhibition, Control, Luciferase, Construct, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Functional Relevance of the Conserved DNA-binding Domain of STAT2

doi: 10.1074/jbc.m500426200

Figure Lengend Snippet: FIG. 4. Chromatin DNA binding ac- tivity of IFN-inducible STAT2:1 het- erodimers is reduced in cells express- ing VV-II STAT2. A, nuclear extracts from cells either left untreated () or treated () for 15 min with 5 ng/ml IFN alfacon-1 were incubated with 32P-labeled ISRE element probe. Untreated and IFN- treated nuclear extracts from human fi- brosarcoma HT-1080 cells were used as a positive control. Complexes were resolved by native gel electrophoresis and visual- ized by autoradiography. B, similar to A, but extracts were incubated with 32P-la- beled pIRE probe to assess IFN-inducible ISGF3-independent STAT2:1 hetero- dimer formation and DNA binding. C, chromatin binding activity of complexes containing either intact STAT2 or the VV-II STAT2. Cells were untreated () or treated () with 5 ng/ml IFN alfacon-1 for 30 min. Following cell lysis and DNA son- ication, aliquots were collected and used as input samples. ChIPs were then per- formed when anti-STAT2 antibody was either added or omitted, as indicated. The precipitated chromatin was analyzed us- ing primers specific for a 6–16 ISRE and an IRF-1 GAS. Primers for -actin were used to confirm the immunoprecipitation is specific for STAT2. D, the signal inten- sity of each band was determined. Histo- grams representing signal intensity ra- tios of each ChIP sample band to its corresponding input band for the 6–16 and IRF-1 primer sets are provided. Data are representative of three independent experiments.

Article Snippet: Antibodies against phospho-STAT2 and phospho-STAT1 were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Binding Assay, Incubation, Labeling, Positive Control, Nucleic Acid Electrophoresis, Autoradiography, Activity Assay, Lysis, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Hes1 attenuates type I IFN responses via VEGF-C and WDFY1

doi: 10.1084/jem.20180861

Figure Lengend Snippet: Hes1 deficiency results in enhanced TLR3-induced expression of type I IFN and ISGs. (A) Heatmap of superinduced genes by poly(I:C) in Hes1 fl/fl Cre-ER T2 (Hes1 KO) BMDMs versus Hes1 +/+ Cre-ER T2 (WT) cells. BMDMs were stimulated without or with 1 µg/ml poly(I:C) for the indicated periods. Ifnb1 in listed genes is highlighted in red. FC, fold change. (B) Percentage of ISGs (defined on the Interferome database) among all superinduced genes by poly(I:C) at 3 h in Hes1 KO BMDMs. (C) qPCR analysis of ISG mRNA in WT and Hes1 KO BMDMs stimulated with poly(I:C) for 3 h. (D) qPCR analysis of Mx1 in WT and Hes1 KO BMDMs stimulated with IFN-β (10 U/ml) for 3 h. (E) Immunoblotting analysis of phosphorylated (p-) and total STAT1 (Tyr701) and STAT2 (Tyr689) in whole-cell lysates of WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (top lanes). Levels of p38α served as loading controls. (F) qPCR analysis of Ifnb1 in WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (left). Cumulative results of Ifnb1 expression are shown (right). (G) qPCR analysis of Ifnb1 and Mx1 in WT and Hes1 KO BMDMs stimulated with various concentrations of poly(I:C) (horizontal axes) for 2 h. (H) ELISA of IFN-β in supernatant of WT and Hes1 KO BMDMs stimulated with poly(I:C) for various times (horizontal axes). Data are representative of one (A and B) or three independent experiments (C–E, F [left], and G; mean + SD of technical triplicates in C, D, F [left], and G) or are pooled from three (F [right] and H; mean ± SD in H) independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

Article Snippet: Antibodies against STAT2 (1:1,000, 72604), p-STAT1 (1:1,000, 7649), p-ERK (1:1,000, 9101), ERK (1:1,000, 9102), p-p38 (1:1,000, 9215), p-p65 (1:1,000, 3033), p65(1:1,000, 4764), IκBα (1:1,000, 4812), TBK1 (1:1,000, 3013), p-JNK (1:1,000, 9251), p-TBK1 (1:1,000, 5483), p-IKKε (1:1,000, 8766), IKKε (1:1,000, 3416), and p-IRF3 (1:500, 4947) were purchased from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Brazilian Journal of Infectious Diseases

Article Title: Antiviral and myocyte protective effects of IL-28A in coxsackievirus B3-induced myocarditis

doi: 10.1016/j.bjid.2014.10.007

Figure Lengend Snippet: IL-28A increased the expression of STAT1 and STAT2 in heart tissue. Uninfected and CVB3-infected mice were treated with or without IL-28A (40 μg/kg) for 4 days, then the heart tissues of infected mice were removed on day 4 post-infection and subjected to Western blot analysis. Actin was used as a loading control. The amount of STAT2, STAT1 and Actin was quantified using NIH Image J and the ratio of STAT2 or STAT1 to Actin was graphed in the lower panels. IL-28A treatment resulted in a significant increase in the expression of STAT1 and STAT2 compared to infected group (** p < 0.01).

Article Snippet: The membranes were blocked in 5% BSA for 40 min at room temperature and then incubated individually at 4 °C overnight with primary antibodies, including rabbit polyclonal antibodies against STAT1 (1:1000) and actin (1:2000), and mouse monoclonal antibodies against STAT2 (1:200) (Cell Signaling Technology, Inc., Danvers, MA).

Techniques: Expressing, Infection, Western Blot, Control

Journal: ACS Nano

Article Title: Echinacoside-Zinc Nanomaterial Inhibits Skin Glycation by Suppressing the Transcriptional Activation of the Receptor for Advanced Glycation End-Products

doi: 10.1021/acsnano.3c04726

Figure Lengend Snippet: PPZn inhibited RAGE transcriptional activation via MDM2. (A) CHX chase assay for the half-life of RAGE. Western blot representative images and quantitative analysis of MDM2, RAGE, and β-actin protein levels in HaCaT cells after the indicated treatment. (B) Correlation analysis between MDM2 and AGER in skin was performed using the GEPIA2 web server. (C) Western blot representative images and quantitative analysis of MDM2, RAGE, and β-actin protein levels in HaCaT cells after the indicated treatment. (D) qRT-PCR detection of RAGE and MDM2 mRNA levels in HaCaT cells after the indicated treatment. (E) Relative luciferase activity of RAGE promoter in HaCaT cells after the indicated treatment by dual luciferase reporter gene assay. (F) Western blot representative images and quantitative analysis of MDM2, and β-actin protein levels in HaCaT cells after the indicated treatment. (G) Flowchart of SM pulldown. (H, I) GO (H) and KEGG (I) enrichment analysis results of SM pulldown-enriched proteins. All values are presented as the mean ± SD; P -values determined by two-sided Student’s t test. * P < 0.05, ** P < 0.01, compared with oe. MDM2.

Article Snippet: Then, HaCaT cells were washed and blocked before incubation with primary antibodies against MDM2 (Proteintech, 1:200) and STAT2 (Zenbio, 1:100) at 4 °C overnight.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Reporter Gene Assay

Journal: ACS Nano

Article Title: Echinacoside-Zinc Nanomaterial Inhibits Skin Glycation by Suppressing the Transcriptional Activation of the Receptor for Advanced Glycation End-Products

doi: 10.1021/acsnano.3c04726

Figure Lengend Snippet: MDM2 formed a transcriptional complex by interacting with STAT2 to promote the transcriptional activation of RAGE. (A) Results of screening for MDM2 interacting transcription factors by Venn diagram. (B) Correlation analysis between STAT2 and AGER in the skin was performed using the GEPIA2 web server. (C, D) Relative luciferase activity of a RAGE promoter in HaCaT cells after the indicated treatment by a dual luciferase reporter gene assay. (E) qRT-PCR detection of RAGE mRNA levels in HaCaT cells after the indicated treatment. (F) Western blot representative images and quantitative analysis of MDM2, STAT2, RAGE, and β-actin protein levels in HaCaT cells after the indicated treatment. (G) Co-IP assays in HEK-293T cells after the indicated treatment. All values are presented as the mean ± SD; P -values determined by two-sided Student’s t test. ** P < 0.01, compared with the control; ## P < 0.01, compared with oe. MDM2.

Article Snippet: Then, HaCaT cells were washed and blocked before incubation with primary antibodies against MDM2 (Proteintech, 1:200) and STAT2 (Zenbio, 1:100) at 4 °C overnight.

Techniques: Activation Assay, Luciferase, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, Control

Journal: ACS Nano

Article Title: Echinacoside-Zinc Nanomaterial Inhibits Skin Glycation by Suppressing the Transcriptional Activation of the Receptor for Advanced Glycation End-Products

doi: 10.1021/acsnano.3c04726

Figure Lengend Snippet: PPZn suppressed RAGE expression by inhibiting the transcriptional complex MDM2/STAT2. (A) Molecular docking of MDM2 (blue) and STAT2 (yellow). (B) Biacore analysis of ECH-Zn bonds to MDM2, STAT2, STAT2 600–750 , and STAT2 Δ600–750 . (C) Representative images of PLA assays of HaCaT cells with MDM2 and RAGE interaction after the indicated treatment. Scale bar, 5 μm. (D) ECH-Zn cellular uptake was detected using coumarin-6 fluorescent dye. Representative images of IF in HaCaT cells after the indicated treatment. Scale bar, 50 μm. (E) Representative IF images of mouse skin tissue after the indicated treatment. Scale bar, 50 μm. (F) Representative images of IHC of mouse skin tissue after the indicated treatment are shown in a. Meanwhile, the statistical results of the relative IHC staining index are shown in b. Scale bar, 50 μm. All values are presented as the mean ± SD; P -values determined by two-sided Student’s t test. * P < 0.05, ** P < 0.01, compared with model.

Article Snippet: Then, HaCaT cells were washed and blocked before incubation with primary antibodies against MDM2 (Proteintech, 1:200) and STAT2 (Zenbio, 1:100) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry