KIR2DL1 alleles *00401 and *010 showed different signaling intensity and inhibition of lipid raft polarization at immune synapse upon stimulation with 721.221-Cw6 cells. YT-Indy cells were retrovirally transduced with KIR2DL1 alleles *00401 and *010 fused with FLAG tag. Similarly expressing cells were collected by flow cytometry cell sorting. YT-Indy cells expressing FLAG-tagged KIR2DL1 alleles *00401 and *010 were stimulated with target 721.221-Cw6 cells at a 1:2 (E/T) ratio for the indicated times. The stimulated cells were lysed and immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with antibodies to (A) SHP-2 and (B) β-arrestin 2. Membranes were stripped and reprobed with FLAG antibody for loading control. (C) Positive and negative are representatives of lipid raft polarization and inhibition of lipid raft polarization, respectively. 2DL1 indicates cells stained with KIR2DL1 antibody, GM1 for lipid raft, and BF for conjugate formation. Calculated percentages of lipid raft polarization in YT-Indy/KIR2DL1*00401401 and YT-Indy/KIR2DL1*010010 in the presence of 721.221-Cw6 are shown in the lower panel. Images were obtained by a Nikon Eclipse E800 fluorescence microscope. The experiment was repeated 3 times. **P < .05. Error bars represent SD.