human trail‐r2‐fc chimeric protein  (Enzo Biochem)

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A Mice were infected with LCMV, and Il15 transcript levels were measured in the indicated organs 24 h postinfection. Data are represented as fold induction after normalization to levels in corresponding naïve tissue. Data indicate mean ± SEM of n = 3 mice per group and show one representative of two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. B–E Mice were infected with LCMV, and flow cytometry was applied on splenic NK cells from infected animals to assess frequencies of IL‐15Rβ‐positive cells (B), IL‐15Rβ expression levels (C), AKT phosphorylation (D), or S6 phosphorylation (E). For AKT and S6 phosphorylation, representative histograms and cumulative results are depicted. MFI, mean fluorescence intensity; Isotype, isotype‐matched control antibody. Data indicate mean ± SEM of n = 3 (B–D) or n = 4 (E) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05. F, G Splenic NK cells from naïve donors were stimulated in vitro for 1 h with IL‐15, and phosphorylation of AKT (F) or S6 (G) was measured. Data indicate mean ± SEM of n = 3 (F) or n = 4 (G) mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. H GZMB expression was measured in splenic NK cells after stimulation with IL‐15 for 20 h. Data indicate mean ± SEM of n = 4 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using one‐way ANOVA with Tukey post‐test. *** P < 0.001; **** P < 0.0001. I WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and GZMB expression was measured in NK cells. Values shown were normalized to unstimulated controls. Data show n = 3 mice per group and show one representative of at least three independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Infection, Two Tailed Test, Flow Cytometry, Expressing, Fluorescence, In Vitro, Cell Culture

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: A NK cells were MACS‐purified from single‐splenocyte suspensions from naïve donors, stimulated in vitro for 1 h with IL‐15, and phosphorylation of S6 was measured by flow cytometry. One representative of two independent experiments is depicted ( n = 1 mouse per group). B AKT and S6 phosphorylation were measured in naive splenic NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using unpaired two‐tailed t ‐test. C Splenocytes were cultured with IL‐15 ± wortmannin (WRM)/LY294002, and GZMB expression was measured in NK cells. Data shown are mean ± SEM of n = 3 mice per group from at least two independent experiments. Statistical analyses were performed using one‐way ANOVA with Dunn's post‐test. * P < 0.05; ** P < 0.01. D, E WT splenocytes were cultured with IL‐15 ± TRAIL‐R2‐Fc chimeric protein, and AKT phosphorylation (D) or S6 phosphorylation (E) was measured in NK cells. Values shown were normalized to unstimulated control. Data show n = 5 mice per group, pooled from two independent experiments. Statistical analyses were performed using paired two‐tailed t ‐test. * P < 0.05; ** P < 0.01. F–H Splenocytes from naïve WT and Trail −/− mice were cultured with the indicated cytokines, and frequencies of IFNγ + NK cells (F), or IFNγ‐expression levels in NK cells (G) were measured after 5 h. Alternatively, naïve splenocytes were cultured with IL‐18/IL‐12, and IFNγ was measured in the supernatant at the indicated time points (H). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of two (F, G) or three (H) independent experiments. Unpaired two‐tailed t ‐test was used. * P < 0.05. I, J MACS‐purified DX5 + cells were cultured in wells coated with an anti‐NK1.1 antibody, and frequencies of IFNγ + NK cells were measured after 5 h (I) or GZMB expression was measured after 24 h (J). Data shown are mean ± SEM of n = 3–4 mice per group and are representative of three (I) or one (J) independent experiments. Unpaired two‐tailed t ‐test was used. ** P < 0.01. K–O Flow cytometry was applied on NK‐92 cells to assess expression of TRAIL (K) and the TRAIL receptors DR4 (L) and DR5 (M). NK‐92 cells were stimulated with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation (N) or GZMB expression (O) was measured by flow cytometry ( n = 1 per condition). (N, O) Data show one representative of 2 independent experiments.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Purification, In Vitro, Flow Cytometry, Two Tailed Test, Cell Culture, Expressing

Journal: EMBO Reports

Article Title: Non‐apoptotic TRAIL function modulates NK cell activity during viral infection

doi: 10.15252/embr.201948789

Figure Lengend Snippet: Human peripheral blood mononuclear cells were cultured with IL‐2 ± human TRAIL‐R2‐Fc chimeric protein, and S6 phosphorylation was measured in CD56 bright NK cells. Data were pooled from three independent experiments ( n = 9 donors per condition). Values shown were normalized to unstimulated controls. Representative histograms are also shown (right‐hand side). Statistical analyses were performed using Wilcoxon matched‐pairs signed rank test. ** P < 0.01.

Article Snippet: TRAIL signaling was blocked using 3 μg/ml of human TRAIL‐R2‐Fc chimeric protein (Enzo Life Sciences).

Techniques: Cell Culture