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: Frontiers in Immunology

Article Title: Hypothermia Promotes Interleukin-22 Expression and Fine-Tunes Its Biological Activity

doi: 10.3389/fimmu.2017.00742

Figure Lengend Snippet: Effects of hypothermic preconditioning on interleukin (IL)-22 bioactivity. Epithelial (-like) IL-22-responsive human DLD1 (A) and Caco2 colon (B) carcinoma cells as well as HepG2 hepatoma cells (C) were preincubated for 6 h without additional stimulation at 30 or 37°C. Thereafter, cells maintained under the respective ambient temperatures were further kept as unstimulated control or stimulated with IL-22 (20 ng/ml). After 1 or 3 h, cells were harvested and IL-22-induced signal transducer and activator of transcription (STAT)-3 activation was determined by immunoblot analysis for pSTAT3. (A–C) Densitometric quantification of experiments ( n = 3) is depicted as raw data (means ± SD). ** P < 0.01, *** P < 0.001 compared to unstimulated control at the respective temperature and time point; # P < 0.05, ## P < 0.01, ### P < 0.001; statistical analysis for individual time points, one-way ANOVA with post hoc Bonferroni correction. (D) One representative of the three independently performed experiments (for each cell type) is shown. (E–H) DLD1 cells were preincubated for 6 h without additional stimulation at 30 or 37°C. (E) Thereafter, cells were further kept under the respective ambient temperatures as unstimulated control or were stimulated with IL-22 (20 ng/ml). After the indicated time periods, α1ACT mRNA expression was determined by real-time polymerase chain reaction (PCR) using GAPDH for normalization. Data are shown as means ± SD [1 h, n = 5 (30 and 37°C); 4 h, n = 8 (30°C) and n = 7 (37°C); 12 h, n = 8 (30 and 37°C); 24 h, n = 6 (30°C), and n = 4 (37°C)]; * P < 0.05, ** P < 0.01; statistical analysis on fold-induction at each time point, unpaired Student’s t -test. AUC, area under the curve. (F) Thereafter, cells were further kept under the respective ambient temperatures as unstimulated control or stimulated with interferon (IFN)γ (20 ng/ml). After 1 or 3 h, cells were harvested and IFNγ-induced STAT1 activation was determined by immunoblot analysis for pSTAT1. One representative of three independently performed experiments is shown. Densitometric quantification of these experiments ( n = 3) is depicted in (G) as raw data (means ± SD). ** P < 0.01, *** P < 0.001 compared to unstimulated control at the respective temperature and time point; ## P < 0.01; statistical analysis for individual time points, one-way ANOVA with post hoc Bonferroni correction. (H) Thereafter, cells were further kept under the respective ambient temperatures as unstimulated control or stimulated with IFNγ (20 ng/ml). After 4 or 12 h, IL-18 binding protein (IL-18BP) mRNA expression was determined by real-time PCR using GAPDH for normalization. Data are shown as means ± SD (4 h, n = 4; 12 h, n = 5); * P < 0.05, ** P < 0.01 compared to unstimulated control of the respective temperature and time point; # P < 0.05; statistical analysis on raw data for individual time points, one-way ANOVA with post hoc Bonferroni correction.

Article Snippet: Antibodies: total STAT3-#124H6 (mouse monoclonal antibody); total STAT1, pSTAT1-Y701-#D4A7 (both rabbit polyclonal antibodies); pSTAT3-Y705-#D3A7 (rabbit monoclonal antibody); all from Cell Signaling, Frankfurt, Germany.

Techniques: Control, Activation Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Binding Assay