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Proteintech mouse monoclonal anti stat2 antibody
Mouse Monoclonal Anti Stat2 Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+monoclonal+anti+stat2+antibody/STAT2+Antibody/pm37741128-73-28-32
Average 94 stars, based on 82 article reviews

mouse monoclonal anti stat2 antibody - by Bioz Stars, 2026-10

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Blocking Assay:

Article Title: Silencing SIRT1 promotes the anti-HBV action of IFN-α by regulating Pol expression and activating the JAK-STAT signaling pathway.
Article Snippet: Purpose: The purpose this study is to investigate the impact of SIRT1 on the anti-HBV activity of IFN-α and further elucidate its underlying mechanism.. Methods: HepG2.2.15 cells stably transfected with HBV virus were chosen as the primary study subject.. IFN-α was used to stimulate the cells and regulate the expression of SIRT1, and the JAK-STAT pathway and HBV-related indices were measured by qRT-PCR, Western blotting and ELISA.

Incubation:

Staining:

Image Search Results

Figure 7. ESM1 promoter reporters are transactivated by STAT1::STAT2. (A) A diagram shows the relative positions of full-length (FL) and fragments of ESM1 promoter reporters. (B) Responses of the FL reporter, and (C) the individual fragments of ESM1 promoter to STAT1 and STAT2 or the HDD mutants were investigated. (D) Reporter assays of the P3 fragment of the ESM1 promoter containing two mutated Binding elements (BEs) as indicated. (E) A schematic illustrates the relative positions of qPCR probes to putative BEs for ChIP-qPCR experiments. (F) Antibody-pulled-down chromatins were analyzed by qPCR. Rb, rabbit. TSS, transcription start site. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Journal: International journal of molecular sciences

Article Title: IRF5 Mediates Artery Inflammation in Salt-Sensitive Hypertension by Regulating STAT1 and STAT2 Phosphorylation to Increase ESM1 Transcription: Insights from Bioinformatics and Mechanistic Analysis.

doi: 10.3390/ijms26083722

Figure Lengend Snippet: Figure 7. ESM1 promoter reporters are transactivated by STAT1::STAT2. (A) A diagram shows the relative positions of full-length (FL) and fragments of ESM1 promoter reporters. (B) Responses of the FL reporter, and (C) the individual fragments of ESM1 promoter to STAT1 and STAT2 or the HDD mutants were investigated. (D) Reporter assays of the P3 fragment of the ESM1 promoter containing two mutated Binding elements (BEs) as indicated. (E) A schematic illustrates the relative positions of qPCR probes to putative BEs for ChIP-qPCR experiments. (F) Antibody-pulled-down chromatins were analyzed by qPCR. Rb, rabbit. TSS, transcription start site. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: For antigen retrieval, the sections were placed in citrate solution and heated at 37 ◦C for 10 min. After retrieval, the slides were rinsed three times with PBS (pH 7.4) for 5 min each in the shaker and then incubated overnight at 4 ◦C with primary antibodies: rabbit monoclonal IRF5 (#ab181553, 1:200, Abcam, Cambridge, UK), mouse monoclonal STAT1 (#sc-464, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and mouse monoclonal STAT2 (#sc-166201, 1:100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Techniques: Binding Assay, ChIP-qPCR

Figure 8. Central illustration. High salt stimulation upregulates IRF5 expression, which enhances the phosphorylation and dimerization of STAT1 and STAT2. The activated STAT1/STAT2 complex translocates into the nucleus, binds to the ESM1 promoter region, and promotes ESM1 transcription. Elevated ESM1 expression contributes to vascular remodeling and the development of salt-sensitive hypertension, as illustrated by the transition from a physiological to a pathological vascular pheno- type. P, phosphorylation.

Journal: International journal of molecular sciences

doi: 10.3390/ijms26083722

Figure Lengend Snippet: Figure 8. Central illustration. High salt stimulation upregulates IRF5 expression, which enhances the phosphorylation and dimerization of STAT1 and STAT2. The activated STAT1/STAT2 complex translocates into the nucleus, binds to the ESM1 promoter region, and promotes ESM1 transcription. Elevated ESM1 expression contributes to vascular remodeling and the development of salt-sensitive hypertension, as illustrated by the transition from a physiological to a pathological vascular pheno- type. P, phosphorylation.

Techniques: Expressing, Phospho-proteomics

pI215L colocalizes with STAT2 at the nucleus. (A) hSTAT2-FLAG interacts with pI215L in BSRT7 cells. BSRT7 cells were co-transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and with pIRES-I215L-myc (2 μg/1 × 10 6 cells). 24 h post-transfection, cells were stimulated with Universal Type I IFN (500 U/mL) for 1 h. Cells were then collected and processed for immunoprecipitation with an anti-myc antibody and analyzed by Western blot labeling with antibodies against FLAG to detect STAT2, against myc to detect pI215L and against actin. (B) pI215L colocalizes with STAT2 in the cell nucleus. Vero cells were transfected with EV or pIRES-I215L-myc (2 μg/1 × 10 6 cells) expression plasmids. After 24 h, cells were stimulated with IFN-I (250 U/mL) for 1 h. After treatment, cells were fixed and stained with DAPI (blue), anti-STAT2 (green) and anti-myc (I215L; red) antibodies and examined by a confocal microscope. Merged images of the different channels are also shown.

Journal: Frontiers in Microbiology

Article Title: African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation

doi: 10.3389/fmicb.2022.1081035

Figure Lengend Snippet: pI215L colocalizes with STAT2 at the nucleus. (A) hSTAT2-FLAG interacts with pI215L in BSRT7 cells. BSRT7 cells were co-transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and with pIRES-I215L-myc (2 μg/1 × 10 6 cells). 24 h post-transfection, cells were stimulated with Universal Type I IFN (500 U/mL) for 1 h. Cells were then collected and processed for immunoprecipitation with an anti-myc antibody and analyzed by Western blot labeling with antibodies against FLAG to detect STAT2, against myc to detect pI215L and against actin. (B) pI215L colocalizes with STAT2 in the cell nucleus. Vero cells were transfected with EV or pIRES-I215L-myc (2 μg/1 × 10 6 cells) expression plasmids. After 24 h, cells were stimulated with IFN-I (250 U/mL) for 1 h. After treatment, cells were fixed and stained with DAPI (blue), anti-STAT2 (green) and anti-myc (I215L; red) antibodies and examined by a confocal microscope. Merged images of the different channels are also shown.

Article Snippet: Monoclonal mouse antibodies anti-STAT2 (B-3, sc-514,193), anti-b-actin (C-4, sc-47,778) and the secondary antibody anti-m-IgGk (BP-HRP, sc-516,102) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Labeling, Expressing, Staining, Microscopy

pI215L induces STAT2 ubiquitination and degradation. (A) STAT2 and pSTAT2 levels were analyzed in presence of increasing concentrations of pIRES-I215L-myc (0.25, 1, or 2.5 μg/1 × 10 6 cells) or EV (2.5 μg/1 × 10 6 cells) in HEK-293 by Western blot assay. Antibodies against STAT2, pSTAT2, myc and actin were employed. STAT2, pSTAT2, and pI215L-myc levels were quantified according with their actin levels and relativized with the EV control using ImageJ. The dose effect of pI215L-myc on STAT2 was quantified as STAT2/Myc. (B) Graphical representation of STAT2 versus pSTAT2 levels from the quantification of the Western blot analyzed samples corresponding to (A) . (C) STAT2 mRNA levels were analyzed in COS-1 cells transfected with EV (2.5 μg/1 × 10 6 cells) or increasing concentrations of pIRES-I215L (0.5 or 2.5 μg/1 × 10 6 cells) in absence or in presence of IFN-I by qRT-PCR. (D) I215L mRNA levels were amplified by qRT-PCR in the same conditions than (C) to verify the correct transcription of I215L in COS-1 transfected cells. All data reflect mean ± SEM ( n = 3). Data were statistically analyzed by using a Student t test (ns, not significant). (E) STAT2 levels were analyzed in presence or absence of the proteasome inhibitor MG132 (10 μM) in pIRES-I215L-myc or EV HEK-293 T transfected cells (2 μg/1 × 10 6 cells) by Western blot assay. Antibodies against STAT2, myc and actin were employed. STAT2 levels were quantified according with their actin levels and relativized with the corresponding EV control using ImageJ. (F) Immunoprecipitation of STAT2 in COS-1 cells transfected with EV or pIRES-I215L-myc. COS-1 cells were co-transfected with pCI-His-hUbiquitin (1 μg/1 × 10 6 cells), hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either EV or pIRES-I215L-myc plasmids (2 μg/1 × 10 6 cells). At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 2 h. Cells were collected and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2, anti-myc and anti-actin antibodies is shown.

Journal: Frontiers in Microbiology

Article Title: African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation

doi: 10.3389/fmicb.2022.1081035

Figure Lengend Snippet: pI215L induces STAT2 ubiquitination and degradation. (A) STAT2 and pSTAT2 levels were analyzed in presence of increasing concentrations of pIRES-I215L-myc (0.25, 1, or 2.5 μg/1 × 10 6 cells) or EV (2.5 μg/1 × 10 6 cells) in HEK-293 by Western blot assay. Antibodies against STAT2, pSTAT2, myc and actin were employed. STAT2, pSTAT2, and pI215L-myc levels were quantified according with their actin levels and relativized with the EV control using ImageJ. The dose effect of pI215L-myc on STAT2 was quantified as STAT2/Myc. (B) Graphical representation of STAT2 versus pSTAT2 levels from the quantification of the Western blot analyzed samples corresponding to (A) . (C) STAT2 mRNA levels were analyzed in COS-1 cells transfected with EV (2.5 μg/1 × 10 6 cells) or increasing concentrations of pIRES-I215L (0.5 or 2.5 μg/1 × 10 6 cells) in absence or in presence of IFN-I by qRT-PCR. (D) I215L mRNA levels were amplified by qRT-PCR in the same conditions than (C) to verify the correct transcription of I215L in COS-1 transfected cells. All data reflect mean ± SEM ( n = 3). Data were statistically analyzed by using a Student t test (ns, not significant). (E) STAT2 levels were analyzed in presence or absence of the proteasome inhibitor MG132 (10 μM) in pIRES-I215L-myc or EV HEK-293 T transfected cells (2 μg/1 × 10 6 cells) by Western blot assay. Antibodies against STAT2, myc and actin were employed. STAT2 levels were quantified according with their actin levels and relativized with the corresponding EV control using ImageJ. (F) Immunoprecipitation of STAT2 in COS-1 cells transfected with EV or pIRES-I215L-myc. COS-1 cells were co-transfected with pCI-His-hUbiquitin (1 μg/1 × 10 6 cells), hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either EV or pIRES-I215L-myc plasmids (2 μg/1 × 10 6 cells). At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 2 h. Cells were collected and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2, anti-myc and anti-actin antibodies is shown.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Transfection, Quantitative RT-PCR, Amplification, Immunoprecipitation, Magnetic Beads, Labeling

The ubiquitin-conjugating catalytic domain of I215L is not required for interaction with STAT2 but is required for its ubiquitination and degradation. (A) Immunoprecipitation of I215L-WT or I215L-C85A in BSRT7 cells co-transfected with hSTAT2-FLAG and with either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc, using an anti-myc antibody. BSRT7 cells were co-transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc (2 μg/1 × 10 6 cells) plasmids. At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 1 h. Then, cells were collected and lysed for immunoprecipitation assay with A/G magnetic beads using anti-myc antibody. Western blot labeling anti-STAT2, anti-myc and anti-actin antibodies is shown. (B) STAT2 levels were analyzed in presence of increasing concentrations of either I215L-C85A-myc or I215L-WT-myc protein (0.25, 1, or 2.5 μg/1 × 10 6 cells) or EV (2.5 μg/1 × 10 6 cells) in HEK-293 by Western blot assay. Antibodies against STAT2, myc and actin were used. STAT2 and pI215L-myc levels were quantified according with their actin levels and relativized with the EV control using ImageJ. (C) Immunoprecipitation of STAT2 in Vero cells transfected with either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc. Vero cells were co-transfected with pCI-His-hUbiquitin (1 μg/1 × 10 6 cells), hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc (2 μg/1 × 10 6 cells) plasmids. At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 2 h. Cells were collected and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2, anti-myc, and anti-actin antibodies is shown.

Journal: Frontiers in Microbiology

Article Title: African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation

doi: 10.3389/fmicb.2022.1081035

Figure Lengend Snippet: The ubiquitin-conjugating catalytic domain of I215L is not required for interaction with STAT2 but is required for its ubiquitination and degradation. (A) Immunoprecipitation of I215L-WT or I215L-C85A in BSRT7 cells co-transfected with hSTAT2-FLAG and with either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc, using an anti-myc antibody. BSRT7 cells were co-transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc (2 μg/1 × 10 6 cells) plasmids. At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 1 h. Then, cells were collected and lysed for immunoprecipitation assay with A/G magnetic beads using anti-myc antibody. Western blot labeling anti-STAT2, anti-myc and anti-actin antibodies is shown. (B) STAT2 levels were analyzed in presence of increasing concentrations of either I215L-C85A-myc or I215L-WT-myc protein (0.25, 1, or 2.5 μg/1 × 10 6 cells) or EV (2.5 μg/1 × 10 6 cells) in HEK-293 by Western blot assay. Antibodies against STAT2, myc and actin were used. STAT2 and pI215L-myc levels were quantified according with their actin levels and relativized with the EV control using ImageJ. (C) Immunoprecipitation of STAT2 in Vero cells transfected with either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc. Vero cells were co-transfected with pCI-His-hUbiquitin (1 μg/1 × 10 6 cells), hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and either pIRES-I215L-WT-myc or pIRES-I215L-C85A-myc (2 μg/1 × 10 6 cells) plasmids. At 24 h post-transfection, cells were treated with IFN-I (500 U/mL) for 2 h. Cells were collected and lysed for STAT2 immunoprecipitation assay with A/G magnetic beads. Western blot labeling with anti-ubiquitin, anti-STAT2, anti-myc, and anti-actin antibodies is shown.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Magnetic Beads, Western Blot, Labeling, Control

$pI215L interacts with STAT2 and promotes its ubiquitination during infection in PAMs. (A) pI215L-GST interacts with STAT2 from extracts of infected PAMs by a pull-down assay. PAMs were infected with Arm/07/CBM/c2 (1 PFU/cell) harvested at 16 hpi and processed for the pull-down experiment with glutathione sepharose beads bound to pI215L-GST or to GST alone as a negative control. Bead-bound proteins were detected by Western blotting labeling with anti-STAT2 and anti-GST antibodies. (B) hSTAT2-FLAG-pI215L interaction during ASFV infection. COS-1 cells were transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and infected with Arm/07/CBM/c2 (1 PFU/cell) for 16 h. Cells were then collected and processed for immunoprecipitation with an anti-FLAG antibody and analyzed by Western blot analysis labeling with antibodies against FLAG to detect STAT2, against pI215L, against pS273R (negative control of co-inmunoprecipitation) and against actin. (C) PAMs were mock infected or infected with Armenia/07/CBM/c2 (1 PFU/cell). At 5 or 15 hpi, cells were untreated or treated with universal type I IFN (250 U/mL). After 1 h of treatment, cells were fixed and stained with DAPI (blue), anti-pI215L (green) and anti-p32 (red) antibodies and examined by a confocal microscope. Individual and merged images of the different channels are shown. (D,E) Nuclear fractionation of mock-infected or infected PAMs at 16 hpi in presence (D) or in absence (E) of IFN-I (500 U/mL). PAMs were seeded in p60 plates and were mock infected (−) or infected (+) with Armenia/07/CBM/c2 strain (1 PFU/cell) for 16 h. At 15 hpi, cells were untreated or treated with type I IFN (500 U/mL) for 1 h. Then, cells were collected and nuclear fractionation was performed. The whole cell extract (WCE), cytoplasmic fraction (S2) and nuclear chromatin fraction (P3) were analyzed by 10% SDS-PAGE, followed by immunoblotting with anti-STAT2 and anti-I215L antibodies. As controls of the fractionation, antibody against nuclear lamin B1 and antibody against cytoplasmic actin were used.$

Journal: Frontiers in Microbiology

Article Title: African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation

doi: 10.3389/fmicb.2022.1081035

Figure Lengend Snippet: pI215L interacts with STAT2 and promotes its ubiquitination during infection in PAMs. (A) pI215L-GST interacts with STAT2 from extracts of infected PAMs by a pull-down assay. PAMs were infected with Arm/07/CBM/c2 (1 PFU/cell) harvested at 16 hpi and processed for the pull-down experiment with glutathione sepharose beads bound to pI215L-GST or to GST alone as a negative control. Bead-bound proteins were detected by Western blotting labeling with anti-STAT2 and anti-GST antibodies. (B) hSTAT2-FLAG-pI215L interaction during ASFV infection. COS-1 cells were transfected with hSTAT2-FLAG (0.4 μg/1 × 10 6 cells) and infected with Arm/07/CBM/c2 (1 PFU/cell) for 16 h. Cells were then collected and processed for immunoprecipitation with an anti-FLAG antibody and analyzed by Western blot analysis labeling with antibodies against FLAG to detect STAT2, against pI215L, against pS273R (negative control of co-inmunoprecipitation) and against actin. (C) PAMs were mock infected or infected with Armenia/07/CBM/c2 (1 PFU/cell). At 5 or 15 hpi, cells were untreated or treated with universal type I IFN (250 U/mL). After 1 h of treatment, cells were fixed and stained with DAPI (blue), anti-pI215L (green) and anti-p32 (red) antibodies and examined by a confocal microscope. Individual and merged images of the different channels are shown. (D,E) Nuclear fractionation of mock-infected or infected PAMs at 16 hpi in presence (D) or in absence (E) of IFN-I (500 U/mL). PAMs were seeded in p60 plates and were mock infected (−) or infected (+) with Armenia/07/CBM/c2 strain (1 PFU/cell) for 16 h. At 15 hpi, cells were untreated or treated with type I IFN (500 U/mL) for 1 h. Then, cells were collected and nuclear fractionation was performed. The whole cell extract (WCE), cytoplasmic fraction (S2) and nuclear chromatin fraction (P3) were analyzed by 10% SDS-PAGE, followed by immunoblotting with anti-STAT2 and anti-I215L antibodies. As controls of the fractionation, antibody against nuclear lamin B1 and antibody against cytoplasmic actin were used.

Techniques: Ubiquitin Proteomics, Infection, Pull Down Assay, Negative Control, Western Blot, Labeling, Transfection, Immunoprecipitation, Staining, Microscopy, Fractionation, SDS Page

Schematic representation of the model of action of pI215L inhibiting the JAK/STAT pathway via STAT2 degradation. On the right side, the activation of the JAK/STAT signaling pathway upon IFN-I stimulus in a non-infected cell is depicted: STAT1 and STAT2 are phosphorylated, together with IRF9 conform the ISGF3 complex, which translocates to the nucleus to act as a transcription factor to induce ISGs transcription. The right side shows the activation of the JAK/STAT pathway in an ASFV-infected cell, where the viral protein I215L interacts with STAT2 in the nucleus, promoting its ubiquitination and degradation, thus preventing ISGF3 complex formation and therefore the transcription of the genes involved in the response to IFN-I. STAT2 degradation by the proteasome has been depicted in the cytoplasm, although the hypothesis that the proteasomal machinery translocates into the nucleus to carry out the degradation of ubiquitinated STAT2 (dashed arrow), cannot be excluded. Biorender program was used for the realization of this illustration.

Journal: Frontiers in Microbiology

Article Title: African swine fever virus ubiquitin-conjugating enzyme pI215L inhibits IFN-I signaling pathway through STAT2 degradation

doi: 10.3389/fmicb.2022.1081035

Figure Lengend Snippet: Schematic representation of the model of action of pI215L inhibiting the JAK/STAT pathway via STAT2 degradation. On the right side, the activation of the JAK/STAT signaling pathway upon IFN-I stimulus in a non-infected cell is depicted: STAT1 and STAT2 are phosphorylated, together with IRF9 conform the ISGF3 complex, which translocates to the nucleus to act as a transcription factor to induce ISGs transcription. The right side shows the activation of the JAK/STAT pathway in an ASFV-infected cell, where the viral protein I215L interacts with STAT2 in the nucleus, promoting its ubiquitination and degradation, thus preventing ISGF3 complex formation and therefore the transcription of the genes involved in the response to IFN-I. STAT2 degradation by the proteasome has been depicted in the cytoplasm, although the hypothesis that the proteasomal machinery translocates into the nucleus to carry out the degradation of ubiquitinated STAT2 (dashed arrow), cannot be excluded. Biorender program was used for the realization of this illustration.

Techniques: Activation Assay, Infection, Ubiquitin Proteomics

a EM density at contour level of 1.81 shown in transparent surface. Proteins are depicted as cartoon models. b Schematic diagram showing the key residues for STAT1-STAT2 dimer, and close-up view of T404 in STAT1-STAT2 interface. c MS/MS spectra for the phosphorylated chymotryptic peptide pTLVEQRSGGSGKGSNKGPLGVTEELHIISF. This quadropoly charged peptide has an observed m/z of 795.1537 Da and is within −0.38 ppm of the expected mass. This spectrum is dominated by H 3 PO 4 loss, consistent with the presence of a phosphorylated S or T residue. The masses of the b 3 and b 5 ions are consistent with phosphorylation at T404. d STAT2-null U6A cells expressing WT, T404A, or T404E STAT2 were treated with IFN-β (100 IU/mL) for 30 min or untreated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2. e STAT1-null U3A cells expressing WT, F77A, F172A, Q340W, or Y701F STAT1 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT1, and analyzed by western blot. f U3A cells expressing WT, F77A, F172A, Q340W, or Y701F STAT1 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Total RNAs were analyzed by qRT-PCR. g U6A cells expressing WT, T404A, T404E, F173A, F175A, or Y690F STAT2 were treated with IFN-β (100 IU/mL) for 4 h. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2 or IRF9 and analyzed by western blot. h U6A cells expressing WT, T404A, T404E, F173A, F175A, or Y690F STAT2 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Total RNAs were analyzed by qRT-PCR. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (mutants vs WT, *** P < 0.001).

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: a EM density at contour level of 1.81 shown in transparent surface. Proteins are depicted as cartoon models. b Schematic diagram showing the key residues for STAT1-STAT2 dimer, and close-up view of T404 in STAT1-STAT2 interface. c MS/MS spectra for the phosphorylated chymotryptic peptide pTLVEQRSGGSGKGSNKGPLGVTEELHIISF. This quadropoly charged peptide has an observed m/z of 795.1537 Da and is within −0.38 ppm of the expected mass. This spectrum is dominated by H 3 PO 4 loss, consistent with the presence of a phosphorylated S or T residue. The masses of the b 3 and b 5 ions are consistent with phosphorylation at T404. d STAT2-null U6A cells expressing WT, T404A, or T404E STAT2 were treated with IFN-β (100 IU/mL) for 30 min or untreated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2. e STAT1-null U3A cells expressing WT, F77A, F172A, Q340W, or Y701F STAT1 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT1, and analyzed by western blot. f U3A cells expressing WT, F77A, F172A, Q340W, or Y701F STAT1 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Total RNAs were analyzed by qRT-PCR. g U6A cells expressing WT, T404A, T404E, F173A, F175A, or Y690F STAT2 were treated with IFN-β (100 IU/mL) for 4 h. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2 or IRF9 and analyzed by western blot. h U6A cells expressing WT, T404A, T404E, F173A, F175A, or Y690F STAT2 were treated with IFN-β (100 IU/mL) for 4 h or untreated. Total RNAs were analyzed by qRT-PCR. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (mutants vs WT, *** P < 0.001).

Article Snippet: Mouse monoclonal antibodies against STAT2 (Santa Cruz Biotechnology), IRF9 (ISGF3γ) (Santa Cruz Biotechnology), VSV-G (Santa Cruz Biotechnology), Tyr 701-phosphorylated STAT1 (Cell Signaling), and Tyr 690-phophorylated STAT2 (Cell Signaling), STAT1 (Upstate), were used for western blot analyses.

Techniques: Tandem Mass Spectroscopy, Residue, Phospho-proteomics, Expressing, Western Blot, Quantitative RT-PCR

a U6A cells expressing WT or T404A STAT2 were treated with IFN-α or IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by qRT-PCR. b U6A cells expressing WT or T404A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. A gene ontology (GO) analysis was performed of genes differentially expressed between U6A cells expressing T404A or WT STAT2, stimulated with IFN-β for 4, 8, or 24 h. c The occupancy of the ADAR1 and MX1 promoters by IRF9 in U6A cells expressing WT, T404A, or T404E STAT2, treated with IFN-β (100 IU/mL), was assayed by ChIP, using anti-IRF9. d Samples from c were analyzed by ChIP-seq. The top 100 segments of each sample were used as inputs. Motifs were discovered by MEME v5.0.3. e U6A cells expressing GFP-tagged WT, T404A, or T404E STAT2 were treated with IFN-β (100 IU/mL) for 0, 5, or 15 min. Whole-cell lysates were used for GFP pull-down and were analyzed by western blot. f Working model shows that, T404 phosphorylation, by destabilizing the U-STAT1-U-STAT2 anti-parallel dimer, increases the affinity of STAT2 for activated IFNAR1, expedites the tyrosine phosphorylation of both STAT2 and STAT1, and enhances the affinity of ISGF3 for ISREs. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (*** P < 0.001).

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: a U6A cells expressing WT or T404A STAT2 were treated with IFN-α or IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by qRT-PCR. b U6A cells expressing WT or T404A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. A gene ontology (GO) analysis was performed of genes differentially expressed between U6A cells expressing T404A or WT STAT2, stimulated with IFN-β for 4, 8, or 24 h. c The occupancy of the ADAR1 and MX1 promoters by IRF9 in U6A cells expressing WT, T404A, or T404E STAT2, treated with IFN-β (100 IU/mL), was assayed by ChIP, using anti-IRF9. d Samples from c were analyzed by ChIP-seq. The top 100 segments of each sample were used as inputs. Motifs were discovered by MEME v5.0.3. e U6A cells expressing GFP-tagged WT, T404A, or T404E STAT2 were treated with IFN-β (100 IU/mL) for 0, 5, or 15 min. Whole-cell lysates were used for GFP pull-down and were analyzed by western blot. f Working model shows that, T404 phosphorylation, by destabilizing the U-STAT1-U-STAT2 anti-parallel dimer, increases the affinity of STAT2 for activated IFNAR1, expedites the tyrosine phosphorylation of both STAT2 and STAT1, and enhances the affinity of ISGF3 for ISREs. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (*** P < 0.001).

Techniques: Expressing, Quantitative RT-PCR, ChIP-sequencing, Western Blot, Phospho-proteomics

a HEK293T cells were co-transfected with IKK-ε and STAT2 constructs as indicated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2, and analyzed by western blot. b The indicated samples from an in vitro kinase assay were analyzed by western blot. Kinases were tagged with MYC, and STAT2 were tagged with Flag as substrate. c Purified Flag-STAT2 was mixed with commercial IKK-ε, immunoprecipitated with anti-Flag, then immunoblotted with anti-IKK-ε. d HEK293T cells were co-transfected with IKK-ε and STAT2 as indicated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2, and analyzed by western blot. e HME cells were pretreated with amlexanox (2 μM) for 30 min, followed by IFN-β (100 IU/mL) for 4 h. Total RNA was analyzed by qRT-PCR. f Whole-cell lysates from HME cells with expression of shRNAs targeting IKK-ε were analyzed by western blot. g HME cells with expression of shRNAs targeting IKK-ε were treated with IFN-β (100 IU/mL) for 4 h. Total RNA was analyzed by qRT-PCR. h BJ (primary human foreskin fibroblasts) cells were infected with VSV (MOI = 1) for 1, 3 or 6 h. Whole-cell lysates were analyzed by western blot. i Hela cells were transfected with poly I:C (10 ng/mL) for 1 h, or 3 h. Whole-cell lysates were analyzed by western blot. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (*** P < 0.001).

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: a HEK293T cells were co-transfected with IKK-ε and STAT2 constructs as indicated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2, and analyzed by western blot. b The indicated samples from an in vitro kinase assay were analyzed by western blot. Kinases were tagged with MYC, and STAT2 were tagged with Flag as substrate. c Purified Flag-STAT2 was mixed with commercial IKK-ε, immunoprecipitated with anti-Flag, then immunoblotted with anti-IKK-ε. d HEK293T cells were co-transfected with IKK-ε and STAT2 as indicated. Whole-cell lysates were used for immunoprecipitations of Flag-STAT2, and analyzed by western blot. e HME cells were pretreated with amlexanox (2 μM) for 30 min, followed by IFN-β (100 IU/mL) for 4 h. Total RNA was analyzed by qRT-PCR. f Whole-cell lysates from HME cells with expression of shRNAs targeting IKK-ε were analyzed by western blot. g HME cells with expression of shRNAs targeting IKK-ε were treated with IFN-β (100 IU/mL) for 4 h. Total RNA was analyzed by qRT-PCR. h BJ (primary human foreskin fibroblasts) cells were infected with VSV (MOI = 1) for 1, 3 or 6 h. Whole-cell lysates were analyzed by western blot. i Hela cells were transfected with poly I:C (10 ng/mL) for 1 h, or 3 h. Whole-cell lysates were analyzed by western blot. Data are shown as means ± SEM from three independent experiments. P values were calculated using the paired ratio t -test (*** P < 0.001).

Techniques: Transfection, Construct, Western Blot, In Vitro, Kinase Assay, Purification, Immunoprecipitation, Quantitative RT-PCR, Expressing, Infection

a Volcano plots for differential gene expression following IFN-β treatment (6 h) of bone marrow-derived macrophages (BMDMs). WT/WT treated vs untreated (left), T403A/T403A treated vs WT/WT treated (right). Transcripts with fold changes > 2 and P < 0.01 after FDR adjustment were defined as differentially expressed genes (DEGs) and are highlighted with blue for down-regulated and red for up-regulated DEGs. b Heatmap of the top 40 DEGs in IFN-β-treated (6 h) BMDMs derived from WT/WT or T403A/T403A mice. c Primary MEFs with WT or T403A STAT2 were treated with 10 IU/mL IFN-β for 2 h, then infected with VSV-GFP (MOI = 1) for 2 h, the expression of VSV G protein was analyzed by the western blot at 12 h post infection. d Primary MEFs with WT or T403 STAT2 were infected with WT VSV (MOI = 1) for 2, 4, 8, or 12 h, and the amounts of VSV genomic RNA (VSV-N) were analyzed by qRT-PCR. e Eight-week-old WT/WT, STAT2 T403A/WT, or STAT2 T403A/T403A mice were infected intravenously with 1 × 10 7 pfu of VSV for 12 days, and their survival was recorded ( n = 20). P < 0.0001 for the difference between WT/WT and T403A/T403A. f Mice from the experiment in c were weighed daily ( n = 20 mice per cohort per dose). Values represent average scores of overall weight loss compared with initial body mass. g Eight-week-old WT/WT, T403A/WT, or T403A/T403A mice were infected intravenously with 8 × 10 5 pfu of HSV for 12 days, and their survival was recorded ( n = 16). P < 0.0001 for the difference between WT/WT and T403A/T403A. h WT/WT and T403A/T403A mice ( n = 4) were infected with VSV for 4, 8, or 12 h and the amount of VSV genomic RNA in the spleens was analyzed by qRT-PCR. i ISG induction in the spleens from f was analyzed by qRT-PCR. Data are shown as means ± SEM from two independent experiments. P values were calculated using the paired ratio t -test (** P < 0.01; *** P < 0.001, ns, not significant).

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: a Volcano plots for differential gene expression following IFN-β treatment (6 h) of bone marrow-derived macrophages (BMDMs). WT/WT treated vs untreated (left), T403A/T403A treated vs WT/WT treated (right). Transcripts with fold changes > 2 and P < 0.01 after FDR adjustment were defined as differentially expressed genes (DEGs) and are highlighted with blue for down-regulated and red for up-regulated DEGs. b Heatmap of the top 40 DEGs in IFN-β-treated (6 h) BMDMs derived from WT/WT or T403A/T403A mice. c Primary MEFs with WT or T403A STAT2 were treated with 10 IU/mL IFN-β for 2 h, then infected with VSV-GFP (MOI = 1) for 2 h, the expression of VSV G protein was analyzed by the western blot at 12 h post infection. d Primary MEFs with WT or T403 STAT2 were infected with WT VSV (MOI = 1) for 2, 4, 8, or 12 h, and the amounts of VSV genomic RNA (VSV-N) were analyzed by qRT-PCR. e Eight-week-old WT/WT, STAT2 T403A/WT, or STAT2 T403A/T403A mice were infected intravenously with 1 × 10 7 pfu of VSV for 12 days, and their survival was recorded ( n = 20). P < 0.0001 for the difference between WT/WT and T403A/T403A. f Mice from the experiment in c were weighed daily ( n = 20 mice per cohort per dose). Values represent average scores of overall weight loss compared with initial body mass. g Eight-week-old WT/WT, T403A/WT, or T403A/T403A mice were infected intravenously with 8 × 10 5 pfu of HSV for 12 days, and their survival was recorded ( n = 16). P < 0.0001 for the difference between WT/WT and T403A/T403A. h WT/WT and T403A/T403A mice ( n = 4) were infected with VSV for 4, 8, or 12 h and the amount of VSV genomic RNA in the spleens was analyzed by qRT-PCR. i ISG induction in the spleens from f was analyzed by qRT-PCR. Data are shown as means ± SEM from two independent experiments. P values were calculated using the paired ratio t -test (** P < 0.01; *** P < 0.001, ns, not significant).

Techniques: Gene Expression, Derivative Assay, Infection, Expressing, Western Blot, Quantitative RT-PCR

a WT/WT, T403A/WT, and T403A/T403A mice ( n = 4) were infected with VSV for 5 days and VSV titers were measured. b Differential pathology of WT/WT and T403A/T403A mice in response to VSV. Shown are brain sections stained with hematoxylin and eosin from the mice described in a . Scale bar, 200 μm. c , d WT or STAT2 T403A mice were injected intravenously with 1 × 10 7 pfu of VSV or buffer. Four days post infection, the brains were excised and analyzed by flow cytometry. e Plasma from c were analyzed by ELISA. f Brains from c were analyzed by qRT-PCR. Data are shown as means ± SEM from two independent experiments. P values were calculated using the paired ratio t -test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant).

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: a WT/WT, T403A/WT, and T403A/T403A mice ( n = 4) were infected with VSV for 5 days and VSV titers were measured. b Differential pathology of WT/WT and T403A/T403A mice in response to VSV. Shown are brain sections stained with hematoxylin and eosin from the mice described in a . Scale bar, 200 μm. c , d WT or STAT2 T403A mice were injected intravenously with 1 × 10 7 pfu of VSV or buffer. Four days post infection, the brains were excised and analyzed by flow cytometry. e Plasma from c were analyzed by ELISA. f Brains from c were analyzed by qRT-PCR. Data are shown as means ± SEM from two independent experiments. P values were calculated using the paired ratio t -test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant).

Techniques: Infection, Staining, Injection, Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

To initiate a systematic antiviral immune response, virus-infected cells produce IFN-I (Left, “Primed-State”), along with induction of STAT2 T404 phosphorylation, which engages an enhanced IFN response to eradicate viruses and the infected cells more efficiently (Middle, “Engaged-State”). Unlike infected cells, distant uninfected cells will be informed by IFN-I to be on guard against potential virus infection (Right, “Informed-State”), but without excessive response.

Journal: Cell Research

Article Title: A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

doi: 10.1038/s41422-020-0386-6

Figure Lengend Snippet: To initiate a systematic antiviral immune response, virus-infected cells produce IFN-I (Left, “Primed-State”), along with induction of STAT2 T404 phosphorylation, which engages an enhanced IFN response to eradicate viruses and the infected cells more efficiently (Middle, “Engaged-State”). Unlike infected cells, distant uninfected cells will be informed by IFN-I to be on guard against potential virus infection (Right, “Informed-State”), but without excessive response.

Techniques: Virus, Infection, Phospho-proteomics

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