Fig. 4. BFA blocks intracellular sorting of Tf from IgA. This biochemical sorting assay is fully described in the Materials and Methods. Briefly, cells were incubated basolaterally with HRP-Tf for 30 minutes at 37°C, then placed on ice and incubated with HRP-Tf, ¹²⁵I-IgA and ¹²⁵I-Tfn for 60 minutes. Excess ligand was rinsed away, and cells were incubated in HRP-Tf in the absence or presence of 10 µM BFA for 15 minutes, and then warmed to 37°C. At the indicated times cells were treated with DAB and H₂O₂ and solubilized. (A) SDS-PAGE and autoradiography of ¹²⁵I-Tf and ¹²⁵I-IgA present in the detergent-soluble fraction following internalization of ligands for the indicated periods of time in the absence or presence of 10 µM BFA. (B) The percentage of pre-bound ¹²⁵I-IgA in the detergent-soluble fraction is plotted as a function of time of internalization for control cells (circles), BFA-treated cells (squares) and cells treated with 33 µM nocodazole (triangles). (C) BFA blocks sorting of pre-internalized IgA and Tf. ¹²⁵I-IgA and HRP-Tf were internalized for 5 minutes at 37°C, then non-internalized ¹²⁵I-IgA was removed by acid wash on ice. BFA was then added for 15 minutes (with the cells still on ice) and the cells then either kept on ice (0 minutes) or incubated at 37°C for 15 minutes and analyzed.