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Figure 3


Fig. 3. Dominant-negative forms of Eps15 and dynamin inhibit internalization of Rh-Tf but not ErbB2. SK-BR-3 cells transfected with EGFP–DN-Eps15 (A,C) or HA–DN-dynamin-1 (B,D) were pretreated with GA for 2 hours before binding unlabeled anti-ErbB2 antibodies (A,C) or Fl-anti-ErbB2 antibodies (B,D). Cells were then warmed for 30 minutes with Rh-Tf, fixed and permeabilized. (A,B) EGFP–DN-Eps15 (A, green) or HA–DN-dynamin (B, blue) are shown with Rh-TF and ErbB2 in deconvolved images, each from a z-stack of a field in which one cell expressed DN-Eps15 (A) or DN-dynamin (B). ErbB2 was detected with Alexa-Fluor-350 goat anti-mouse antibodies (A, blue; C) or by fluorescein fluorescence (B, green; D). Scale bar: 10 µm. (C,D) Internalization of ErbB2 and Rh-Tf in cells expressing EGFP–DN-Eps15 (C) or HA–DN-dynamin (D) (+), and untransfected cells on the same coverslips (–). Cells showing at least three intracellular punctae were scored positive. Numbers shown are averages of two experiments (counting at least 100 transfected and 100 untransfected cells in each experiment), the data from which varied by <10%.





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