phosphorylated stat2 y690  (Cell Signaling Technology Inc)

Journal: Microbial Cell Factories

Article Title: Secretion of functional interferon by the type 3 secretion system of enteropathogenic Escherichia coli

doi: 10.1186/s12934-024-02397-y

Figure Lengend Snippet: Bacterially secreted IFN YNS induces activation of the IFN-1 pathway. Cells were incubated with supernatants of bacterial cultures, washed, lysed, and their protein extracts were subjected to SDS-PAGE and western blot analysis using antibodies against phosphorylated STAT2 (phospho-STAT) and actin (loading control). Cells incubated with commercial IFNβ or IFNα (2 nM) were used as positive controls, while a sample of untreated cells was used as a negative control. A HeLa cells were incubated with supernatants collected from cultures of WT EPEC, Δ escN , and Δ sepD strains in the presence or absence of a plasmid encoding for IFN YNS (pIFN) that were grown aerobically or B anaerobically. C HT-29 (left) and Caco-2 (right) cells were incubated with supernatants from cultures of EPEC Δ sepD and EPEC Δ sepD that express and secrete IFN YNS (pIFN). D Caco-2 cells were incubated with supernatants from a culture of EPEC Δ sepD + pIFN and with commercial IFNα2 (0.5 nM), alone or following pre-incubation with an anti-IFNα2 antibody (5 nM) for 1 h. IFNβ (0.5 nM) was used as a positive control, and untreated cells and cells incubated with supernatant from a culture of EPEC Δ sepD were used as negative controls

Article Snippet: The following primary antibodies were used: rabbit anti-phosphorylated STAT2 (Abcam Inc.), diluted 1:600; rabbit anti-IFNα2 (Abcam), diluted 1:1000; rabbit anti-JNK1 + JNK2 + JNK3 antibody (Abcam Inc.), diluted 1:1000; mouse anti-DnaK (Abcam, Inc.), diluted 1:5,000, and mouse anti-Tir, which is a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot, Negative Control, Plasmid Preparation, Positive Control

Journal: Microbial Cell Factories

Article Title: Secretion of functional interferon by the type 3 secretion system of enteropathogenic Escherichia coli

doi: 10.1186/s12934-024-02397-y

Figure Lengend Snippet: The addition of IFN YNS -secreting bacteria to host cells induces IFN-1 pathway activation. WT EPEC, Δ escN , and Δ sepD strains in the presence or absence of a plasmid-encoding human IFN (pIFN) were added to the HeLa cells and co-cultured for 3 h. The cells were then washed and lysed, and their protein extracts were subjected to SDS-PAGE and western blot analysis using anti-phosphorylated STAT2 (phospho-STAT) ( A ) or anti-JNK ( B ) and anti-actin antibodies (loading control). JNK and its degradation fragments are indicated on the right of the gel

Article Snippet: The following primary antibodies were used: rabbit anti-phosphorylated STAT2 (Abcam Inc.), diluted 1:600; rabbit anti-IFNα2 (Abcam), diluted 1:1000; rabbit anti-JNK1 + JNK2 + JNK3 antibody (Abcam Inc.), diluted 1:1000; mouse anti-DnaK (Abcam, Inc.), diluted 1:5,000, and mouse anti-Tir, which is a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada).

Techniques: Bacteria, Activation Assay, Plasmid Preparation, Cell Culture, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: SARS-CoV-2 ORF7a Mutation Found in BF.5 and BF.7 Sublineages Impacts Its Functions

doi: 10.3390/ijms25042351

Figure Lengend Snippet: SARS-CoV-2 ORF7a: H47Y mutant is ineffective in antagonizing IFN-I response. ( A ) H47Y mutation abrogates ORF7a’s ability to block IFN-I response. ISG56-promoter driven firefly luciferase reporter levels upon infection with SARS-CoV-2 WT/H47Y or empty vector (E.V.) in the presence or absence of human IFN-β. Data were analyzed by normalizing firefly luciferase to renilla luciferase activities and then normalizing to non-IFN-β-treated samples to obtain fold induction. E.V. was set to 100-fold induction. ( B ) H47Y mutation decreases ORF7a ubiquitination. Lysates of HEK 293T cells cotransfected with plasmids for ubiquitin and either WT, H47Y ORF7a or empty vector (E.V.) followed by immunoprecipitations using an anti-V5 antibody and immunoblot analysis probing for ORF7a (V5), ubiquitin (HA) and β-actin. ( C ) ORF7a H47Y mutant fails to block STAT2 phosphorylation. STAT2 phosphorylation levels were determined by immunoblotting of lysates of HEK 293T cells transfected with plasmids for either SARS-CoV-2 WT, H47Y mutant ORF7a or empty vector (E.V.) and treated with human IFN-β (5 units/mL). Densitometry analysis of phosphorylated STAT2 over total STAT2 levels (pSTAT2: STAT2) in the presence of IFN-β is shown below (E.V. condition is set at 1). ( D , E ) H47 mutation impairs ORF7a’s ability to suppress host antiviral response upon SARS-CoV-2 infection. Calu-3 cells transfected with plasmids for SARS-CoV-2 WT/H47Y or empty vector (E.V.) were infected with SARS-CoV-2-eGFP/ΔORF7a virus followed by RT-qPCR for viral subgenomic (sg) RNA levels in ( D ) and expression of the indicated ISGs in ( E ). In ( E ), fold expression changes relative to mock-infected and normalized to GAPDH are shown. ( F , G ) SARS-CoV-2 replication in the presence of ORF7a H47Y is severely diminished upon IFN-β treatment. HEK 293T-hACE2 cells transiently expressing either ORF7a WT or H47Y were treated with either IFN-β or mock (PBS) followed by infection with SARS-CoV-2-eGFP/ΔORF7a virus. Viral RNA levels were determined by RT-qPCR and ORF7a expression was analyzed by immunoblotting. In ( F ), SARS-CoV-2 spike copy number normalized to GAPDH in the absence of IFN-β. In ( G ), percentage of normalized SARS-CoV-2 spike copy number was determined after setting mock-treated conditions (for both ORF7a WT and ORF7a H47Y) at 100%. Immunoblot images of ORF7a protein expression are shown. The fold differences between mock- and IFN-β-treated conditions are indicated. Error bars represent means ± S.D for 3 independent experiments. In A, C and G, one sample t -test (two-tailed) was used for comparisons between E.V. and ORF7: WT/H47Y conditions, while unpaired t -test (two-tailed) was used for comparisons between ORF7a WT and H47Y conditions. Comparisons in E and F were performed using unpaired t -test (two-tailed). Representative immunoblot images ( n = 3) are shown. *, p < 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant.

Article Snippet: The following antibodies were used for probing STAT2 and phosphorylated STAT2: anti-STAT2 (D9J7L; Cell Signaling) and anti-phospho-STAT2 (Y690) (D3P2P; Cell Signaling).

Techniques: Mutagenesis, Blocking Assay, Luciferase, Infection, Plasmid Preparation, Ubiquitin Proteomics, Western Blot, Phospho-proteomics, Transfection, Virus, Quantitative RT-PCR, Expressing, Two Tailed Test