Figure 7: Synergistic activation of NF-κB is Vav1-dependent.

(a) Rested NKL cells were stimulated with NKG2D and/or 2B4 in combination with or without CD94 engagement by receptor crosslinking. Lysates were immunoblotted for pS536-p65, pS276-p65, p65, p-Akt, p-Erk1/2, pY174-Vav1 or actin. The normalized intensities of the phosphorylated p65 relative to p65 are presented. (b) Rested NKL-κB-GFP cells were stimulated with plate-immobilized NKG2D and 2B4 in combination with or without CD94 for 6 h. GFP expression in the reporter NKL cells was analysed by flow cytometry, and representative result (left) and statistical bar charts (right) are shown. Values represent mean±s.d. (c) Rested NKL cells transfected with control siRNA or Vav1-specific siRNA were stimulated with NKG2D and/or 2B4 by receptor crosslinking. Lysates were immunoblotted for pS536-p65, pS276-p65, p65, p-Akt, p-Erk1/2, Vav1 or actin. The normalized intensities of the phosphorylated p65 relative to p65 are presented. (d) Rested NKL-κB-GFP cells transfected with control siRNA or Vav1-specific siRNA were 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 (left) and statistical bar charts (right) are shown. Values represent mean±s.d. **P<0.01; ***P<0.001 (two-sided Student’s t-test). Data are representative of at least three independent experiments.