Figure 8.

FcRn-mediated internalization of Fc is not significantly affected by endosome alkalinization. Mouse FcRn-transfected HULEC-5A endothelial cells were preincubated for 15 min with 1 μM bafilomycin and then incubated for 15 min with TxR-WT and Cy5-dextran in the continued presence of bafilomycin. Cells were then fixed for analysis. (A–C) Fluorescence micrographs of bafilomycin-treated cells. (A) Internalization of Cy5-dextran by five cells in the field, (B) Expression of GFP-FcRn by two cells in the field. (C) Internalization of TxR-WT by the two GFP-FcRn-expressing cells. (D) Quantification of internalization of Fc and dextran. For each of the conditions above, total cell fluorescence was quantified for each fluor from projections of 3D volumes. Expression of GFP-FcRn enriches internalization of Fc relative to dextran in both bafilomycin-treated (n = 30, 25) and untreated cells (n = 31, 27). (E) Internalization of Fc increases linearly with expression of GFP-FcRn in bafilomycin-treated cells (n = 23). Scale bar, 10 μm.