Vinculin constitutively associates with talin and Arp2. (A) IL-15– or IL-2–activated splenic NK cells were incubated with ICAM-1 beads, fixed, permeabilized, and stained for WASP or vinculin (green), Arp2/3 (red), and actin (blue). Alexa Fluor 488 secondary antibody was used for WASP and vinculin staining, and Alexa 568 secondary antibody was used for Arp2/3 staining. Actin was stained with Alexa 647–conjugated phalloidin. Images (× 400 magnification) were collected by confocal microscopy as in Figure 1. (B) IL-15– (lane 1) or IL-2–cultured (lane 2) splenic NK cells were lysed. Immunoprecipitation was performed with anti-talin (top), anti-vinculin (center), anti-WASP (bottom), or isotype control antibodies (lane 3) and probed as indicated. Immunoprecipitates were blotted as indicated. Results shown are representative of 3 independent experiments. (C) IL-15– (lane 1) or IL-2–activated (lane 2) splenic NK cells were allowed to bind to ICAM-1–coated plates and then lysed. Immunoprecipitation was performed with anti-talin (top), anti-vinculin (center), or anti-WASP (bottom) and probed as indicated. In the WASP immunoprecipitates in panels B and C, IgG (∼ 50 kD) band is also detected.