Fig. 3. BFA blocks intracellular sorting of Tf from IgA. 3-D images of living, polarized PTR cells were collected in the presence of basolateral Oregon Green-IgA and TxR-Tf after 30 minutes of uptake (A), and following 15 minutes of pretreatment in10 µM BFA and 35 minutes incubation with probes in the continued presence of BFA (B). In control cells, the sorting of IgA from Tf is apparent in the enrichment of IgA (green) in the ARE lacking Tf (red), whereas both IgA and Tf completely codistribute in BFA-treated cells, resulting in a nearly uniform endosome color in each cell. Each field is 40 µm across. (C) Plots of individual pixel intensities from projected image volumes of control and BFA-treated cells, labeled and imaged as described above. Data represent the pooled data of 8 cells from each condition, with data normalized such that the mean IgA and Tf fluorescence are equal. Sorting of IgA is apparent in the enrichment of IgA in the ARE, represented by the population of pixels with a high ratio of IgA/Tf, relative to the common endosomes, whose pixels show a low ratio. In contrast, only a single population of pixels is found in cells treated with BFA.