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Figure S4. Colocalization of internalized αBTX–AChR complexes with fluid-phase and endocytosed Tf. CHO-K1/A5 cells were labeled with Cy3αBTX (BTX; red) for 1 h at 4°C, and the label was chased for 12 h. FITC-dextran (green in top panels; FITC-Dx) or Cy5-Tf (green in bottom panels; TfR) were added to the medium for 5 (left) or 20 min (right) before the end of the incubation. Cy3αBTX-labeled endosomes (open arrowheads) are accessible to a 20-min pulse of FITC-dextran (closed arrowheads), whereas they do not colocalize with a 5-min pulse. This is consistent with the 12-h endosomal structures resembling bonafide late endosomes/lysosomes, which are accessible to fluid-phase cargo from the outside but not to endocytosed TfR. See the lack of colocalization between Cy3αBTX endosomes and Cy5-TfR endosomes in the insets. Insets are magnified images of the areas marked by asterisks; red outline, BTX; green outline, FITC-dextran or Cy5-Tf; white outline, merged image. Bar, 10 µm.