Some PLD1 constitutively localizes to the plasma membrane, especially in GEMs/lipid rafts, and changes FcϵRI distribution in the membrane. (A) Lysates from unstimulated D1/WT cells were fractionated by sucrose density gradient centrifugation, and fractions were analyzed by immunoblotting with anti-FLAG, LAT, and RMCPII Abs. The percentage of the total protein in the indicated fractions was calculated from the densitometric analysis and is the average ± standard deviation from 3 independent experiments. Similar results were obtained when the other cell line of each transfection was examined. (B) FcϵRI distribution in the membrane. Lysates from unstimulated cells presensitized with biotinylated IgE were fractionated by sucrose density gradient centrifugation. Biotinylated IgE was detected with horseradish peroxidase (HRP)-conjugated streptavidin. The percentage of the biotinylated IgE localized to the lipid rafts was calculated from the densitometric analysis and is the average ± standard deviation from 3 different experiments. Statistically significant change compared with the control cells or D1/WT cells is indicated as follows: ^*P < .05. Similar results were obtained when the other cell line of each transfection was examined.