Fig. 6. Co-localization of FcRn–GFP with EEA1 and Rab11 in wortmannin-treated cells. Bar, 5 µm. Sections labeled as apical, tight junctions, nucleus and basolateral were taken 1, 3, 6 and 9 µm, respectively, from the apical surface of the monolayer. Cells were treated with 150 nM wortmannin (A–E) and with 1 µM IgG added to the apical side of the cell monolayer at pH 7.3 (B–E). Inhibition of protein biosynthesis with 25 µg/ml cycloheximide did not prevent the recruitment of FcRn–GFP to vacuoles (data not shown), consistent with the observation in a number of cell types that wortmannin does not affect organelles or transport steps of the biosynthetic pathway (Tuma et al., 1999) and thereby confirming that the apical endocytic pathway is the source of FcRn–GFP found in vacuoles. (A and B) Wortmannin effect on FcRn–GFP distribution in the absence (A) and presence (B) of ligand. (C) EEA1 distribution in cells treated with wortmannin and ligand. (D) Superposition of fluorescence from FcRn–GFP and EEA1 in cells treated with wortmannin and ligand. (E) Superposition of fluorescence from FcRn–GFP and Rab11 in cells treated with ligand and wortmannin. Regions of co-localization appear yellow.