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Fig. 4. Internalization kinetics of ALCAM/CD166 compared to transferrin. (A) ALCAM/CD166 internalization is time dependent and slower than transferrin. Confocal microscopic imaging of IGROV-I cells incubated with I/F8 scFv and anti-FLAG mAb in the presence of transferrin-FITC. The samples were allowed to internalize for the indicated times (in minutes) before acid-stripping of the surface-bound molecules, fixing, permeabilizing and staining with RITC-labeled secondary antibody. Images were acquired sequentially as single transcellular optical sections, and the same section is shown stained with I/F8 (left panels) and with transferrin (right panels). Insets show the non-stripped samples. (B) Internalized ALCAM/CD166 distributes in the sub-membrane region and does not cluster toward the perinuclear area. Confocal microscopic imaging of IGROV-I cells stained with I/F8 plus anti-FLAG mAb, allowed to internalize for 60 minutes, acid-washed to strip the surface if required (right panel), fixed, permeabilized and stained with FITC-labeled secondary antibody. Nuclei were counterstained with Propidium Iodide. Magnification, 600 x.





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