Inhibition of lipid raft microdomains by cholesterol depletion severely attenuates activation of Syk, ERK, and STAT4 in NK cells following FcR + IL-12 stimulation. Purified human NK cells were pretreated with 5 nM MβCD (right column) or DMSO solvent control (Mock Pre-Tx; left column) for 1 hour and then activated via FcγRIIIa ligation in the presence of 10 ng/mL IL-12 for 5 minutes at 37°C. Control conditions consisted of control Ab-treated NK cells (−), NK cells activated via FcR ligation alone (FcR), or NK cells activated with IL-12 alone (IL-12). Cells were lysed and analyzed for p-Syk (A), p-ERK (B), and p-STAT4 (C) by immunoblot analysis. Numbers below immunoblots represent the fold increase in phosphoprotein levels for each condition, normalized to total protein and expressed in relative densitometric units. All results are representative of n = 3 determinations.