Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: iScience

Article Title: RGS2 is an innate immune checkpoint for suppressing Gαq-mediated IFNγ generation and lung injury

doi: 10.1016/j.isci.2025.111878

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Article Snippet: Rabbit Monoclonal anti-phospho-STAT1 (Y701) , Cell Signaling , Cat # 9177S.

Techniques: Recombinant, Protease Inhibitor, Red Blood Cell Lysis, Construct, Control, Software

Journal: Cell Reports

Article Title: MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells

doi: 10.1016/j.celrep.2020.108628

Figure Lengend Snippet: IFN Production Inhibits SARS-CoV-2 Replication (A) Immunoblots of IFNAR1, IL10RB, STAT1, STAT2, IRF9, and actin in Calu-3 cells transfected with indicated pooled siRNA for 48 h are shown. (B) Calu-3 cells were transfected with siRNAs. Forty-eight hours post-transfection, the cells were treated with IFN-β (1,000 U/mL) or IFN-λ (1,700 U/mL) for 8 h prior to RNA extraction. IFIT1 mRNA was quantified using qRT-PCR. Data are expressed as fold change relative to non-treated cells. (C) Calu-3 cells were transfected with siRNAs. At 48 h post-transfection, the cells were infected with SARS-CoV-2 at MOI = 0.125 for a further 48 h. IFIT1 mRNA was quantified using qRT-PCR. Data are expressed as fold change relative to non-treated cells. (D) Viral titer in the collected supernatants were determined by plaque assay in Vero E6 cells. Data show mean ± SD from one representative experiment in triplicate (n = 3) of two independent experiments. (E) The siRNA-transfected Calu-3 cells were infected with SARS-CoV-2 with MOI = 0.125. At 48 h post-infection, cells were fixed, immunostained with rabbit-anti-SARS-CoV-2 NP antibody (green) and DAPI (blue), and imaged using an IC200 high-content imager. Representative immunofluorescence images are shown. Scale bar, 100 μm. (F) The percentage of infection was calculated as the ratio between the number of infected cells stained for SARS-CoV-2 NP and the total amount of cells stained with DAPI. Data are from three independent experiments with three technical replicates.

Article Snippet: Rabbit anti-Phospho STAT1 (Y701) antibody , Cell Signaling , Cat# 9167; RRID: AB_561284.

Techniques: Western Blot, Transfection, RNA Extraction, Quantitative RT-PCR, Infection, Plaque Assay, Immunofluorescence, Staining

Journal: Cell Reports

Article Title: MDA5 Governs the Innate Immune Response to SARS-CoV-2 in Lung Epithelial Cells

doi: 10.1016/j.celrep.2020.108628

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Article Snippet: Rabbit anti-Phospho STAT1 (Y701) antibody , Cell Signaling , Cat# 9167; RRID: AB_561284.

Techniques: Virus, Recombinant, High Molecular Weight, Transfection, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Software

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) IFN-β alone vs media, expressed as ratio of IFN-β/media. (B) Vitamin D alone vs media. (C) IFN-β plus vitamin D vs media alone. Selected targets are grouped into Th1, Th17, and Th2 immune pathways and clusters of IFN-stimulated proteins (ISGs), adhesion molecules, and cytokines controlling homeostatic proliferation, neurotrophic factors, and IL-12 family. Values are fold change in the stimulation condition/media. Intensity of shading shows upregulation (red) or downregulation (blue). Target proteins include BDNF = brain-derived neurotrophic factor; HGF = hepatocyte growth factor; IFN-γ = interferon-γ; IL-2 = interleukin-2; TNF-α = tumor necrosis factor-α; TNFRII = TNF receptor type II; IL-17F, IL-4, IL-5, IL-10, IP-10 = IFN-induced protein-10 (CXCL10); MCP1 = macrophage chemotactic protein-1 (CCL2); I-TAC = IFN-inducible T-cell alpha chemoattractant (CXCL11); LIF = leukemia inhibitory factor; p-Y-STAT1 = phosphotyrosine-STAT1 transcription factor; MxA = myxovirus A; NGF = nerve growth factor; sICAM-1 = soluble intercellular adhesion molecule-1; TPO = thymopoietin; VCAM-1 = vascular cell adhesion molecule; VEGF-α = vascular endothelial growth factor-α, IL-15, IL-7, and IL-12 p40 and IL-12 p70 components.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Derivative Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) In vitro vitamin D enhances IFN-β induction of p-Y-STAT1 in ConA-activated MNCs from 72 untreated patients with MS ( p = 0.00002). (B) p-Y-STAT1 expression on flow cytometry histograms from a representative patient with RRMS-s. The media, IFN, VitD, and IFN+VitD curves are all stained with Alexa Fluor 488–labeled Mab to p-Y-STAT1. (C) MxA protein on Western blot of a representative patient with RRMS-s. ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM Vit D3 (calcitriol) for 12 hours. Gray-scale densities of each band are listed above figure. IFN-β = interferon-β; MxA = myxovirus protein.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: In Vitro, Expressing, Flow Cytometry, Staining, Labeling, Western Blot, Incubation

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: (A) p-Y-STAT1, measured with flow cytometry, is generated in all groups, but there is less induction in SPMS than in HC. (B) MxA protein, measured with Western blots. The trend for enhanced responses in PPMS vs HC was not significant ( p < 0.13, unpaired t test). ConA-activated MNCs were incubated in vitro with 160 U/mL IFN-β-1b for 30 minutes to 48 hours ± preincubation with 200 nM vitamin D3 (calcitriol) for 12 hours. Fold change of vitamin D plus IFN-β compared with IFN-β alone, determined using VDSI: [(IFN + VitD) − (VitD)]/[(IFN) − (no IFN)]; average with p values above SEM bar: * p < 0.05, ** p < 0.01. Comparisons between groups use unpaired t tests, brackets. PPMS = primary progressive MS; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Generated, Western Blot, Incubation, In Vitro

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: In all groups, there is enhanced induction of p-Y-STAT1, measured with flow cytometry. Methods and calculations as in . * p < 0.05, ** p < 0.01. IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β–induced p-Y-STAT1 expression is quantitated with flow cytometry. The median vitamin D level for the entire cohort was 30; low < 30, high ≥ 30 ng/mL 25-OH vitamin D. Methods and calculations as in . * p < 0.05, ** p < 0.01. RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; PPMS = primary progressive MS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Vitamin D enhances responses to interferon-β in MS

doi: 10.1212/NXI.0000000000000622

Figure Lengend Snippet: Vitamin D plus IFN-β (blue line) in most therapy-naive groups had a greater effect than IFN-β alone (yellow line) on activation of STAT1 in ConA-activated lymphocytes and monocytes. Values in radar plot represent median fold change in log 2 scale expression of p-Y-STAT1 measured by flow cytometry. Blue line: (IFN+ VitD) − (Vit D); yellow: (IFN)-(media alone). IFN-β = interferon-β; RRMS-a = active relapsing/remitting MS; RRMS-s = stable RRMS; SPMS = secondary progressive MS.

Article Snippet: Antibodies were primary rat anti-MxA (Biogen), p-STAT1 (Y701) rabbit MAb (Cell Signaling Technology), actin (I-19) goat polyclonal IgG, donkey anti-goat IgG-horseradish peroxidase (HRP), chicken anti-rat IgG-HRP, and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology).

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Structural studies of the IFNλ4 receptor complex using cryoEM enabled by protein engineering

doi: 10.1038/s41467-025-56119-y

Figure Lengend Snippet: A pSTAT1 signaling of IFNω1 (orange), IFNλ3 (blue), and IFNλ4 (red) in Hap1 cells. Curves are fit to a first-order logistic model. Data are presented with mean values and error bars representing ±SEM ( n = 3 biologically independent experiments). B Relative quantification (RQ) of select genes induced by IFNω1, IFNλ3, and IFNλ4 in Hap1 cells treated with saturating concentrations (100 nM) interferon for 6 h as measured by qPCR. Data are presented with mean values. Error bars represent 95% confidence intervals ( n = 3 biologically independent experiments). C Changes in induction of ISG15 or MX1 over time for IFNλ3 and IFNλ4 in Huh7.5.1 cells at a range of concentrations (~0.5, 1.5, 5, and 15 nM). Statistical significance determined by a two-tailed student t -test. Data are presented as mean values ± SD ( n = 3 biologically independent experiments; * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001). D Intracellular HCV genomic RNA level over time following treatment with IFNω1, IFNλ3, or IFNλ4 in Huh7.5.1 cells. Statistical significance was determined by a two-tailed student t -test. Data are presented as mean values ± SD ( n = 3 biologically independent experiments; * = p ≤ 0.05, ** = p ≤ 0.01).

Article Snippet: To quantify activation of pSTAT1, cells were washed thrice with 0.5% PBSA and then stained with an anti-Y701 pSTAT1 monoclonal antibody (Cell Signaling, product #9174S) according to manufacturer instructions.

Techniques: Quantitative Proteomics, Two Tailed Test

Journal: Retrovirology

Article Title: Innate immune defects in HIV permissive cell lines

doi: 10.1186/s12977-016-0275-8

Figure Lengend Snippet: Defects in 3 selected innate immunity pathways in cell lines. a The figure represents a simplified view of the TLR7/TLR8, IFN-gamma and TNF-alpha signaling pathways. Boxes representing genes display the transcriptional levels detected in RNA-seq libraries of resting CD4+ T cells, the four human laboratory cell lines HEK293T, Jurkat, SupT1 and CEM -mock (MO), heat-inactivated (HI) and HIV-infected (HIV)- and 4 samples corresponding to Activated CD4+ T cells at 8 and 24 h after TCR activation. Inset describes the order of the libraries as well as the color-code scale of expression levels (log10 transformation of the number of library size-normalized reads per kilobase of exonic sequence) ranging from 0 ( green ) to ≥2.8 ( red ; lower limit of the 9th-decile of expression values). The expression levels indicated for IFNG and TNF convey the basal expression level before adding the stimuli. b Experimental validation of the functional integrity of the selected innate immunity pathways. The table reports the stimuli applied and the functional read-out measured 24 h after stimulation. A positive sign indicates positive detection of functional read-outs (transcript levels by RT-qPCR or phosphorylation of STAT1 by Western blot analysis). NT not tested, nd not detected

Article Snippet: Total phospho-Stat1 (Mouse anti-P-Y701 Stat1, 1:1000, #612132, BD Biosciences) and Stat1 (Rabbit anti-Stat1 antibody, 1:1000, #9172, Primary Cell Signaling Technology) were detected using standard procedures with Tris-Buffered Saline (TBS)-0.2 % Tween-5 % BSA and PBS-0.2 % Tween-5 % milk as blocking buffers for Phospho-Stat1 and Stat1 respectively.

Techniques: RNA Sequencing Assay, Infection, Activation Assay, Expressing, Transformation Assay, Sequencing, Functional Assay, Quantitative RT-PCR, Western Blot

Journal: Retrovirology

Article Title: Innate immune defects in HIV permissive cell lines

doi: 10.1186/s12977-016-0275-8

Figure Lengend Snippet: Heatmap of expression values of paradigmatic genes involved in antiretroviral defense and signaling relevant to HIV biology. The figure shows the expression values of antiretroviral genes ( APOBEC3G, TRIM5, BST2, MX2, GBP5, and SAMHD1 ) and signaling genes ( JAK, STAT1, IFI16 and STING/TMEM173 ) in RNA-seq libraries of resting CD4+ T cells, cell lines HEK293T, Jurkat, SupT1 and CEM -mock (Mock), heat-inactivated (hiLV) and HIV-infected (LV)- and activated CD4+ T cells at 8 and 24 h after TCR activation . The color-code scale in the inset represents the expression levels (log10 transformation of the number of library size-normalized reads per kilobase of exonic sequence), ranging from green (low) to red (high) expression. Complete hierarchical clustering of genes was based on Pearson correlation of the expression levels. Complete hierarchical clustering of samples was kept as assessed in Additional file : Figure S4. JAK1, JAK2, TMEM173 and GBP5 had a fold change higher than 2 between resting CD4+ T cells and permissive cell lines (Benjamini–Hochberg adjusted p value <0.01, )

Article Snippet: Total phospho-Stat1 (Mouse anti-P-Y701 Stat1, 1:1000, #612132, BD Biosciences) and Stat1 (Rabbit anti-Stat1 antibody, 1:1000, #9172, Primary Cell Signaling Technology) were detected using standard procedures with Tris-Buffered Saline (TBS)-0.2 % Tween-5 % BSA and PBS-0.2 % Tween-5 % milk as blocking buffers for Phospho-Stat1 and Stat1 respectively.

Techniques: Expressing, RNA Sequencing Assay, Infection, Activation Assay, Transformation Assay, Sequencing