Figure 7

Treatment with the PI4K inhibitors wortmannin and quercetin prevents the biosynthetic trafficking of EGFR to the PM. Live cells transiently expressing EGFR-GFP were processed as described for Figure 1, except that wortmannin (200 nM or 10 μM) or quercetin (20 μM) were added 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 ± wortmannin 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. * p < 0.05; *** p < 0.001 (B) Representative confocal images of RBL cells expressing EGFR-GFP either untreated or treated with 200 nM or 10 μM wortmannin from 8 hr time points quantified in A. (C) Flow cytometry was used to calculate the ratio of PM localized EGFR fluorescence to total EGFR fluorescence, and this ratio for each time point ± quercetin is plotted. Error bars indicate ± SE of four independent experiments in which approximately 8,000 EGFR-GFP expressing cells were analyzed at each time point. ** p < 0.01 (D) Representative confocal images of RBL cells expressing EGFR-GFP either untreated or treated with 20 μM quercetin from 8 hr time points quantified in C. Two different cells with 20 μM quercetin shown to represent the range of distributions observed. Scale bars show 5 μm.