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

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 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.

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: Expressing, Phospho-proteomics

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

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.

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

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.

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

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.

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

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.

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: Activation Assay, Infection, Ubiquitin Proteomics