Treatment with PAO inhibits the biosynthetic trafficking of GPI to the PM. Live cells transiently expressing GPI-YFP were processed as described for EGFR expressing cells, PAO (2.5 μM) was added to half of the cells at hour 3. (A) Flow cytometry was used to calculate the ratio of PM localized GPI fluorescence, detected by anti-GFP, to total GPI fluorescence, and this ratio for each time point ± PAO (2.5 μM) is plotted. Error bars indicate ± SD of three independent experiments in which approximately 8,000 GPI-YFP expressing cells were analyzed at each time point, and all time points were normalized to the control ratio at 7 hr, ** p < 0.01. (B) Confocal images of attached RBL cells expressing GPI-YFP either untreated or treated with PAO for 6 hr, as quantified in A. Scale bar shows 5 μm.