Both FcγRIIIa and IL-12R localize to lipid raft domains on NK cells in response to FcR activation in the presence of IL-12. (A) Purified human NK cells were activated via FcγRIIIa ligation in the presence of huIL-12 for 5 minutes at 37°C (column iv). Control conditions consisted of untreated NK cells (column i), NK cells activated via FcR ligation alone (column ii), or NK cells activated with IL-12 alone (column iii). The green channel shows the location of FcγRIIIa and the red channel shows the distribution of IL-12R, while the blue channels represents p-Lck (a component of lipid raft microdomains¹¹ ). Single confocal sections of the cells were captured in multitrack. Each set of frames in a given column is a representative individual NK cell selected from 1 of 20 analyzed cells. Superimposed white-yellow patches signify areas of colocalization of FcγRIIIa IL-12R and p-Lck. (B) Detergent-insoluble cholesterol-enriched lipid rafts were prepared by Brij-98 extraction of purified human NK cells that were left unstimulated (left column) or activated via FcγRIIIa ligation in the presence of IL-12 for 5 minutes (right column) and separated on a sucrose density gradient. A total of 9 sequential fractions collected from the gradient were subjected to immunoblot analysis for p-Lck, FcγRIIIa, and IL-12R. Fractions 2, 3, and 4 correspond to detergent-insoluble lipid fractions; fractions 7, 8, and 9 correspond to detergent-soluble fractions. All results shown are representative of 3 independent experiments.