Treatment with PIK-93 does not significantly affect the biosynthetic trafficking of EGFR to the PM
. Live cells transiently expressing EGFR-GFP were processed as described for Figure 1, except that 1 μM PIK-93 was added to half of the cells at hour 3. (A) Flow cytometry was used to calculate the ratio of PM localized EGFR fluorescence to total EGFR fluorescence, and this ratio for each time point ± PIK-93 is plotted. Error bars indicate ± SE of five independent experiments in which approximately 8,000 EGFR-GFP expressing cells were analyzed at each time point. n.s., not significant (B) Representative confocal images of RBL cells expressing EGFR-GFP either untreated or treated with 1 μM PIK-93, cells are from 8 hr time points quantified in A. Scale bar shows 5 μm. (C) Degranulation was measured for cells pretreated with denoted concentrations of PIK-93 for 40 min before stimulation with antigen (10 μg/ml) for 25 min. Bars represent average degranulation response from two experiments, in which each condition was assayed in triplicate, error bars indicate ± SD.