(A) Rested NKL cells were mixed with S2, S2-ULBP1, S2-CD48, or S2-ULBP1+CD48 insect cells, as indicated. After incubation for 20 or 40 min, Vav1 was immunoprecipitated from lysates and immunoblotted for p-Tyr and pY160-Vav1. Blots were re-probed for Vav1. The normalized intensities of the phosphorylated Vav1 at tyrosines or at tyrosine 160 relative to total Vav1 are presented.
(B) Granzyme B release assay with rested NKL cells that were transfected with control siRNA or Vav1-specific siRNA and incubated with S2 cells or S2 cells expressing ULBP1 and/or CD48, as indicated. After incubation for 2 hr, granzyme B in the supernatants was measured by ELISA. The increase of granzyme B after incubation with target cells relative to granzyme B upon incubation of NKL cells alone (ΔGranzyme B) is presented. Values represent mean ± SD. Data are representative of at least three independent experiments.