Talin associates with PIPKIγ and is required for enrichment of PIP₂ after binding of LFA-1 to ICAM-1. (A) WT or talin-KO ES-derived NK cells (ES-NK) and WT or WASP-KO IL-15–activated cells were incubated with ICAM-1 (IC-1)– or PLL–coated beads, fixed, permeabilized, stained with anti-PIP₂ and Alexa Fluor 488–conjugated secondary antibody, and analyzed by confocal microscopy as in Figure 1. (B) Fluorescence intensity of PIP₂ staining at the contact site between a cell and a bead coated with ICAM-1 (▭) or PLL (▬) is expressed as a ratio of fluorescence at the contact site over fluorescence at an opposite point. Error bars indicate SEM, n = 10 per condition. *P < .05 and **P < .01 compared with corresponding PLL condition. (C) IL-15–activated splenic NK cells were lysed and immunoprecipitation was performed with anti-PIPKIγ (lane 1) or isotype control antibody (lane 4). Immunoprecipitates were probed with anti PIPKIγ (left) or anti-talin (right). As a positive control 20 μg (lane 2) or 2.5 μg (lane 3) of purified rat brain extract protein containing the 90- and 87-kDa isoforms of PIPKIγ was immunoblotted with anti-PIPKIγ antibody.