Journal: Immunity

Article Title: Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection

doi: 10.1016/j.immuni.2020.07.004

Figure Lengend Snippet:

Article Snippet: Proteins were transferred onto nitrocellulose membranes, blocked with either 5% skim milk (Devondale, Brunswick, Australia) or 5% BSA (for phospho-proteins) in PBS with 0.05% Tween-20 (PBST) for 1 h, and detected using the following primary antibodies: rat anti-caspase-11 (4E11, Enzo Life Sciences), rat anti-caspase-1 (1E11, Enzo Life Sciences), rat anti-MLKL (3H1; available from Merck), rabbit anti-phospho S345 MLKL (EPR9515[2]; Abcam, Cambridge, UK), rat anti-caspase-8 (3B10; Enzo Life Sciences), rabbit anti-cleaved caspase-8 (D5B2; Cell Signaling Technology), rabbit anti-RIPK3 (ProSci, Poway, CA, USA), rabbit anti-cleaved caspase-3 (Asp175; Cell Signaling Technology), rabbit anti-caspase-7 (D2Q3L, Cell Signaling), rabbit anti-caspase-9 (Cell Signaling Technology), rabbit anti-GSDMD (EPR19828, Abcam), mouse anti-PARP (C2-10, Santa Cruz), rat anti-BID (2D1-3, WEHI), mouse anti-HSP70 (BRM-22, Sigma Alrich) and rabbit anti-β-actin-HRP (Cell Signaling Technology).

Techniques: Virus, Recombinant, Protease Inhibitor, Western Blot, Lactate Dehydrogenase Assay, Plasmid Preparation, Sequencing, 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: Nuclear factor of activated T cells (NFAT) signaling in peripheral blood mononuclear cells (PBMC) is mandatory for interleukin (IL)-22 upregulation by anti-CD3/-CD28 upon normothermic or hypothermic culture conditions. (A–C) Where indicated, PBMC were pretreated for 30 min with CsA (A,B) or with FK506 (C) at the specified concentrations. Cells were further kept as unstimulated control or stimulated with anti-CD3 (0.2 µg/ml)/-CD28 (0.02 µg/ml) antibodies at 37 or 33°C. All cultures were adjusted to a final concentration of 0.002% (A,B) or 0.008% DMSO (C) (vehicle for CsA and FK506). After 8 h, IL-22 [ (A) , n = 10; (C) , n = 6] or IL-8 [ (B) , n = 10] mRNA expression was assessed by real-time polymerase chain reaction using GAPDH for normalization. Data are shown as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to unstimulated control; ## P < 0.01, ### P < 0.001; statistical analysis for either 37 or 33°C, one-way ANOVA with post hoc Bonferroni correction. (D) PBMC from five different donors were kept unstimulated or stimulated with anti-CD3 (0.2 µg/ml)/-CD28 (0.02 µg/ml) antibodies at 37 or 33°C for the indicated time periods. Thereafter, CD3 + T-cell isolation was performed followed by immunoblot analysis for detection of nucleic NFAT-c2. (E) Densitometric quantification of experiments shown in Figure D. Data are expressed as means ± SEM. * P < 0.05, ** P < 0.01; statistical analysis, unpaired Student’s t -test.

Article Snippet: Antibodies: NFAT-c2-#4G6-G5, mouse monoclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany); β-actin-#AC15, mouse monoclonal antibody (Sigma-Aldrich).

Techniques: Control, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Cell Isolation, Western Blot