Fig. 2. Localization of internalized ErbB2, Rh-Tf, EGFR and clathrin. SK-BR-3 cells were pretreated with GA for 1 hour before binding Fl-anti-ErbB2 (A,C,E,F) or Fl-anti-EGFR (B,D,G) and warming for 5 minutes (with Rh-Tf in A,B and E) before acid washing and immunofluorescence analysis. (C,D) Clathrin heavy chain (CHC, red) was detected by immunofluorescence. (E-G) High-magnification views of the boxed regions in A,C and D, respectively. Right-hand panels in A-D and bottom panels in E-G show merged images. (A) Epifluorescence images. All other panels show maximum-intensity projections of deconvolved z-stacks. Scale bars: 10 µm (bar in B applies to B-D). (H) Quantification of colocalization of Rh-Tf with ErbB2 or EGFR in cells treated as in A,B, except that internalization was for 2 minutes.