Figure 1: Synergistic activation of NF-κB by NKG2D and 2B4 co-engagement. | Nature Communications

Figure 1: Synergistic activation of NF-κB by NKG2D and 2B4 co-engagement.

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

Figure 1

(a) NKL cells rested in the absence of IL-2 for 24 h were stimulated with NKG2D and/or 2B4 by receptor crosslinking for the indicated time. Cell lysates were immunoblotted with Abs to phospho-p65 at serine 536 (pS536), p65, phospho-IKKα/β at serine 176/180 (pS176/180), IKKβ, phospho-Akt at serine 473 (pS473), phospho-Erk1 and 2, or actin. The normalized intensities of the phosphorylated p65 and IKKα/β relative to their total forms are presented. (b) Rested NKL cells were treated as in a to stimulate NKG2D and 2B4 for the indicated time. Lysates were immunoblotted for phospho-p65, phospho-IKKα/β, phospho-IκBα at serine 32/36 (pS32/36), IκBα, phospho-Erk1/2 or actin. (c) Representative confocal images (top) of conjugates between rested NKL cells loaded with CFSE (green) and P815 target cells as indicated. Conjugates were fixed, permeabilized and stained with 4,6-diamidino-2-phenylindole (DAPI; blue) and mAb to p65, anti-mouse IgG-Biotin followed with Alexa Fluor 647 (red)-Streptavidin. The number beneath the overlay image is the mean nuclear fluorescence intensity (MFI)±s.d. of p65 from ⩾50 NKL-target cell conjugates. Statistical bar charts (bottom) for MFI of p65 in the nucleus are represented as fold change. Values represent mean±s.d. Scale bar, 5 μm. (d) Rested NKL cells were stimulated with plate-immobilized mAbs to NKG2D and/or 2B4 for 1 h. Equal amounts of protein from cytoplasmic and nuclear extracts were immunoblotted with mAb to p65. (e) Nuclear extracts collected as in d were added into a 96-well plate immobilized with double-stranded oligonucleotide containing the consensus NF-κB-binding sequence. The amount of p65 bound to the oligonucleotide was measured by colorimetric assay. Values represent mean±s.d. (f) Rested NKL cells transduced with a κB-GFP reporter construct were stimulated with plate-immobilized mAbs to NKG2D and/or 2B4 for 6 h. GFP expression in NKL-κB-GFP 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