Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

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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: Nature Communications

Article Title: Corticosteroid suppression of antiviral immunity increases bacterial loads and mucus production in COPD exacerbations

doi: 10.1038/s41467-018-04574-1

Figure Lengend Snippet: Fluticasone propionate suppresses TLR3 and RIG-I-induced interferon responses but does not block type I IFN signalling. a – d BEAS-2B cells were treated with fluticasone propionate at 1 and 10 nM concentrations and stimulated with rhinovirus-A1 or receptor-specific agonists. Cell lysates were collected at 24 h post stimulation. a IFNβ and IFNλ2/3 mRNA expression following RV-A1 stimulation was measured by quantitative PCR. b IFNβ and IFNλ2/3 mRNA expression following poly(I:C) stimulation was measured by quantitative PCR. c IFNβ and IFNλ2/3 mRNA expression following RIG-I agonist (transfected 5′ppp-dsRNA) stimulation was measured by quantitative PCR. Transfected dsRNA lacking the 5′ triphosphate was used as a ‘RIG-I control’. d IFNβ and IFNλ2/3 mRNA expression following MDA-5 agonist (transfected HMW Poly(I:C) directly pre-coupled to the transfection reagent LyoVec) stimulation was measured by quantitative PCR. LyoVec without Poly(I:C) (LyoVec control) was used as a control for transfection. e , f BEAS-2B cells were transfected with interferon-β promoter–reporter constructs together with ΔTRIF, ΔMAVS or vector control (pUNO1) and then treated with fluticasone propionate (FP) 1 or 10 nM or medium control 3 h later. Cells were collected 24 h later and relative light units (RLU) were determined. e IFNβ promoter activity following transfection with ΔTRIF. f IFNβ promoter activity following transfection with ΔMAVS. g , h BEAS-2B cells were treated with fluticasone propionate at 1 and 10 nM concentrations and stimulated with recombinant IFN-β. g 2 ′– 5 ′ OAS, viperin and MX1 mRNA expression at 24 h post stimulation was measured by quantitative PCR. h Cell extracts were collected at 1 h post stimulation and analysed by western blotting with antibodies to pSTAT1 Y701, STAT1, pSTAT2 Y690 and STAT2. In a – g , data represent mean (±SEM) comprising three independent experiments, analysed by one-way ANOVA with Bonferroni post test. ns non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001. In h , data shown are representative of results from three independent experiments. i C57BL/6 mice were treated intranasally with fluticasone propionate (20 μg) or vehicle DMSO control and additionally with recombinant 10 4 units IFN-β. Interferon-stimulated genes 2 ′- 5 ′ OAS , and Viperin mRNAs in lung tissue at 8 h post infection were measured by quantitative PCR. Data represents mean (±SEM) of five mice per treatment group, representative of at least two independent experiments. Data were analysed by one-way ANOVA with Bonferroni post test. ns non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following primary antibodies were used: rabbit anti-pSTAT1 Y701 (1:1000 diluted; New England BioLabs), anti-pSTAT2 Y690 (1:1000, New England BioLabs), anti-STAT1 (1:1000 diluted; New England BioLabs) and anti-STAT2 (1:1000 diluted; New England BioLabs).

Techniques: Blocking Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Activity Assay, Recombinant, Western Blot, Infection

Journal: Cell reports

Article Title: A FLCN-TFE3 Feedback Loop Prevents Excessive Glycogenesis and Phagocyte Activation by Regulating Lysosome Activity

doi: 10.1016/j.celrep.2020.01.042

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-pStat1 [Y701] (58D6)-153Eu , Fluidigm , Cat#3153003A; RRID: AB_2811248.

Techniques: Purification, Blocking Assay, Recombinant, Transfection, Flow Cytometry, Magnetic Beads, Lysis, Extraction, SYBR Green Assay, Staining, shRNA, Sequencing, Plasmid Preparation, Software

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