FIGURE 4:

Cross-linking TfR also prevents sorting into recycling tubules. HMEC-1 cells stably expressing HA-FcRn-EGFP were incubated with rat anti-HA antibody (2 μg/ml) (A) or mouse anti–TfR ectodomain antibody (25 μg/ml) alone (C) or together with rat anti-HA antibody (D) for 1 h at 37°C. Samples were washed and further incubated with Alexa 568–human transferrin and Alexa 647–goat anti-rat antibody (A), Alexa 647–human transferrin and Alexa 568–goat anti-rat antibody (B), Alexa 568–human IgG and Alexa 647–goat anti-mouse antibody (C), or Alexa 568-goat anti-rat antibodies alone and Alexa 647–goat anti-mouse antibodies (D) for 15 min at 37°C. Samples were then washed and transferred to an open perfusion chamber for live-cell imaging acquisition, and medium containing 10 μM BFA was added. Time series were recorded immediately after the addition of BFA. Representative confocal middle sections of each condition are shown, with arrowheads indicating examples of tubules containing IgG or transferrin and lacking cross-linked TfR and FcRn multimeric complexes, and displayed as described in the Figure 1 legend. Scale bars, 10 μm.