Figure 3: NKG2D and 2B4 coactivation induces disparate and cooperative signals for NF-κB activation. | Nature Communications

Figure 3: NKG2D and 2B4 coactivation induces disparate and cooperative signals for NF-κB activation.

From: Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition

Figure 3

(a) Rested NKL cells were stimulated with NKG2D and/or 2B4 by receptor crosslinking for the indicated time. Cell lysates were immunoblotted for phospho-p65 at serine 536 (pS536), phospho-p65 at serine 276 (pS276) or p65. The normalized intensities of the phosphorylated p65 relative to total p65 are presented. (b) Rested NKL cells were stimulated with NKG2D and 2B4 for 5 min after pretreatment with PI3K inhibitor (LY294002; 20 μM) and/or MEK inhibitor (PD98059; 20 μM) for 30 min. Lysates were analysed by immunoblotting for the indicated phosphorylations of p65. (c) Cytokine release assays with rested NKL cells after pretreatment with 20 μM LY294002 and/or 20 μM PD98059 for 30 min and then stimulation with NKG2D and 2B4 for 12 h in the presence of the inhibitor. Thereafter, IFN-γ (left) and MIP-1α (right) in the supernatants were measured by ELISA. Values represent mean±s.d. (d) Primary rested NK cells after expansion were stimulated with the indicated receptors by receptor crosslinking for 5 min. Lysates were analysed by immunoblotting as in b. (e) Primary rested NK cells after expansion were stimulated and analysed by immunoblotting as in b. (f) Frequency of NK cells that displayed IFN-γ or TNF-α expression after pretreatment of PBMCs with 20 μM LY294002 and/or 20 μM PD98059 for 1 h and then stimulation with P815 target cells as indicated in the presence of the inhibitor. After incubation for 6 h, cells were stained and analysed by flow cytometry. Representative result (top) and statistical bar charts (bottom) from three experiments are shown. Values represent mean±s.e.m. (g) Rested NKL-κB-GFP cells were pretreated with 20 μM LY294002 and/or 20 μM PD98059 for 1 h and then stimulated with plate-immobilized NKG2D and 2B4 for 6 h. GFP expression in the reporter NKL cells was analysed by flow cytometry, and representative result (top) and statistical bar charts (bottom) are shown. Values represent mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (two-sided Student’s t-test). Data are representative of at least three independent experiments.

Back to article page