Figure 2: NF-κB is required for cytokine and chemokine gene expression by NKG2D and 2B4 coactivation.

(a) Rested NKL cells were pretreated with a NF-κB inhibitor BAY11-7082 at the indicated dose for 1 h and then stimulated with both NKG2D and 2B4 for 8 h. Thereafter, IFN-γ and MIP-1α in the supernatants were measured by ELISA. Values represent mean±s.d. (b) NKL cells were transfected with 300 pmol of control siRNA or siRNA specific for p65. After 24 h, the cells were rested for another 24 h, and lysates were immunoblotted for p65 and actin. (c) Rested NKL cells transfected with control siRNA or p65-specific siRNA were stimulated with NKG2D and/or 2B4 for 3 h. Thereafter, total RNA was prepared from cells, reverse transcribed and the relative mRNA levels of IFN-γ, TNF-α, granzyme B, MIP-1α, MIP-1β and IκBα were determined by real-time PCR and normalized to β-actin mRNA. Values represent mean±s.d. (d) Rested NKL cells transfected with control siRNA or p65-specific siRNA were stimulated as in c for 8 h. IFN-γ and MIP-1α in the supernatants were measured by ELISA. Values represent mean±s.d. (e) Rested NKL cells that were transfected with control siRNA or p65-specific siRNA and stimulated with NKG2D and/or 2B4 for 2 h were used in a granzyme B release assay. Granzyme B in the supernatants was measured by ELISA. Error bars represent the 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.