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: Scientific Reports

Article Title: Comparison of anti-inflammatory and anti-angiogenic effects of JAK inhibitors in IL-6 and TNFα-stimulated fibroblast-like synoviocytes derived from patients with RA

doi: 10.1038/s41598-025-94894-2

Figure Lengend Snippet: ( a ) Western blotting was performed to analyze STAT1, pSTAT1, STAT3, pSTAT3, and β-actin. ( b , c ) Comparison of the ratios of pSTAT1 to STAT1 and pSTAT3 to STAT1. Protein expression was determined by semi-quantitative analysis of digitally captured images. All JAK inhibitors significantly suppressed pSTAT1 and pSTAT3 levels compared to those in the control. Compared to TOF, BAR, PEF, UPA, and FIL suppressed pSTAT1 and pSTAT3 levels. STAT, signal transducer and activator of transcription; JAK, Janus kinase; TOF, tofacitinib; BAR, baricitinib; PEF, peficitinib; UPA, upadacitinib; FIL, filgotinib.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBST at 25 °C for 30 min, incubated with antibodies against anti-STAT1, anti-phosphorylated STAT1 (pSTAT1), anti-STAT3, and anti-pSTAT3 (Cell Signaling Technology, Danvers, MA, US) at 4 °C for 12 h, and further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody at 25 °C for 1 h. The proteins were subsequently visualized using ECL Plus reagent (GE Healthcare Life Sciences, Little Chalfont, UK) on a chemiluminescence analyzer (LAS-3000 mini; Fujifilm, Tokyo, Japan).

Techniques: Western Blot, Comparison, Expressing, Control

Journal: Journal of Interferon & Cytokine Research

Article Title: Type I Interferon-Mediated Induction of Antiviral Genes and Proteins Fails to Protect Cells from the Cytopathic Effects of Sendai Virus Infection

doi: 10.1089/jir.2016.0051

Figure Lengend Snippet: SeV-induced IFN production activates Jak/STAT signaling and ISG induction. (A) U937 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. Nuclear and cytoplasmic protein extracts were prepared and western blots probing for pIRF-3(S386), IRF-3, IRF-7, and IRF-9 were performed. RNA pol II and β-tubulin were the nuclear and cytoplasmic loading controls, respectively. N, nuclear, C, cytoplasmic. (B) U937 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h and whole cell protein lysates were prepared. Western blots probing for pSTAT1(Y701), STAT1(total), pSTAT2(Y689), STAT2(total), ISG15, and IFIT3, and tubulin as the loading control were performed. (C) A549 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h and whole protein lysates were prepared. Western blots probing for pSTAT1(Y701), STAT1(total), pSTAT2(Y689), STAT2(total), IRF-9, IRF-7, and Mx1, and tubulin as the loading control were performed. (D) ISG15 and IFIT3 mRNA expression in U937 cells infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. (E) IRF-7 and Mx1 mRNA expression in A549 cells infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. IRF-7 and Mx1 mRNA induction in (F) primary monocytes and (G) Namalwa cells infected with SeV (150 HA U/mL) for 24 h. All gene induction data are expressed as the % of relative HPRT expression. All data are representative of 3 independent biological replicates. UI, uninfected, untreated. IRF, IFN regulatory factor; ISG, interferon-stimulated gene; HPRT, hypoxanthine-guanine phosphoribosyltransferase.

Article Snippet: The following antibodies and dilutions were used for the western blot analyses: Monoclonal rabbit anti-pIRF-3(S386; Abcam, Cambridge, MA): 1:5,000; monoclonal anti-IRF-3 (Cell Signaling Technologies, Danvers, MA): 1:1,000; polyclonal rabbit anti-IRF-7 (Santa Cruz Biotechnologies, Dallas, TX): 1:1,000; polyclonal rabbit anti-STAT1 (Santa Cruz Biotechnologies): 1:1,000; monoclonal mouse anti-pSTAT1(Y701; BD Transduction Laboratories, San Jose, CA): 1:5,000; polyclonal rabbit anti-STAT2 (Santa Cruz Biotechnologies): 1:1,000; polyclonal rabbit anti-pSTAT2(Y689; End Millipore, Billerica, MA): 1:2,000; monoclonal mouse anti-IRF-9(ISGF3γ; BD Transduction Laboratories): 1:1,000; monoclonal mouse anti-Actin (Cell Signaling Technologies): 1:5,000; polyclonal rabbit anti-β tubulin (Cell Signaling Technologies): 1:2,000; mouse monoclonal RNA polymerase II (Santa Cruz Biotechnologies): 1:1,000; rabbit polyclonal IFIT3 (Covance, Inc., Denver, PA) 1:1,000; rabbit polyclonal ISG15: 1:1,000 (Santa Cruz Biotechnologies); mouse polyclonal Mx1: 1:1,000 (Abnova, Taipei, Taiwan).

Techniques: Infection, Western Blot, Expressing

Journal: Journal of Interferon & Cytokine Research

Article Title: Type I Interferon-Mediated Induction of Antiviral Genes and Proteins Fails to Protect Cells from the Cytopathic Effects of Sendai Virus Infection

doi: 10.1089/jir.2016.0051

Figure Lengend Snippet: Inhibiting canonical type I IFN signaling during SeV infection does not affect SeV-mediated CPE. (A) U937 cells were treated with 1000 U/mL of IFN-α2a, IFN-β, or IFN-ω for 1 h in the presence or absence of the IFNAR2 neutralizing antibody. Western blot analysis was performed to detect phosphorylated STAT1(Y701) and tubulin was used as the loading control. UT: untreated; NA: cells treated with IFNAR2 neutralizing antibody alone; α2a: IFN-α2a treated; α2a + NA: cells treated with both antibody and IFN-α2a; β: IFN-β treated; β + NA: cells treated with both antibody and IFN-β; ω: IFN-ω treated; and ω + NA: cells treated with both antibody and IFN-ω. (B) U937 (panel 1) and A549 cells (panel 2) were infected with SeV (150 HA U/mL) in the presence or absence of IFNAR2 neutralizing antibody. Cell viability was measured 72 h pi. Data are representative of 3 independent biological replicates. Supernatants from cells infected with SeV (150 HA U/mL) for 16 h were inactivated (β-propiolactone) and used to pretreat fresh (C) U937 and (D) A549 cells for 24 h in the presence or absence of the IFNAR or IFNLR neutralizing antibodies. The U937 cells were then infected with either EMCV or SeV (150 HA U/mL) for 72 h and cell viability was measured via MTT assay (U937) or crystal violet assay (A549). Results are shown as the % of cell control. SeV150: virus-inactivated supernatant from SeV-infected cells (150 HA U/mL); SeV150 + IFNAR2 NA: treated with virus-inactivated supernatant from SeV-infected cells (150 HA U/mL) in the presence of IFNAR2-neutralizing antibody; SeV150 + IFNLR NA: treated with virus-inactivated supernatant from SeV-infected cells (150 HA U/mL) in the presence of IFNLR-neutralizing antibody. IFNAR, IFN receptor; IFNLR, IFN-lambda receptor.

Article Snippet: The following antibodies and dilutions were used for the western blot analyses: Monoclonal rabbit anti-pIRF-3(S386; Abcam, Cambridge, MA): 1:5,000; monoclonal anti-IRF-3 (Cell Signaling Technologies, Danvers, MA): 1:1,000; polyclonal rabbit anti-IRF-7 (Santa Cruz Biotechnologies, Dallas, TX): 1:1,000; polyclonal rabbit anti-STAT1 (Santa Cruz Biotechnologies): 1:1,000; monoclonal mouse anti-pSTAT1(Y701; BD Transduction Laboratories, San Jose, CA): 1:5,000; polyclonal rabbit anti-STAT2 (Santa Cruz Biotechnologies): 1:1,000; polyclonal rabbit anti-pSTAT2(Y689; End Millipore, Billerica, MA): 1:2,000; monoclonal mouse anti-IRF-9(ISGF3γ; BD Transduction Laboratories): 1:1,000; monoclonal mouse anti-Actin (Cell Signaling Technologies): 1:5,000; polyclonal rabbit anti-β tubulin (Cell Signaling Technologies): 1:2,000; mouse monoclonal RNA polymerase II (Santa Cruz Biotechnologies): 1:1,000; rabbit polyclonal IFIT3 (Covance, Inc., Denver, PA) 1:1,000; rabbit polyclonal ISG15: 1:1,000 (Santa Cruz Biotechnologies); mouse polyclonal Mx1: 1:1,000 (Abnova, Taipei, Taiwan).

Techniques: Infection, Western Blot, MTT Assay, Crystal Violet Assay

Journal: Immunity

Article Title: The Cytokine TGF-β Induces Interleukin-31 Expression from Dermal Dendritic Cells to Activate Sensory Neurons and Stimulate Wound Itching

doi: 10.1016/j.immuni.2020.06.023

Figure Lengend Snippet:

Article Snippet: Anti-mouse pStat1 , Cell Signaling , Cat# 9171S; RRID: AB_331591.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Reverse Transcription, Gene Expression, Sequencing, Software