Fig. 3. The relationship between lipid rafts and the small- and large-scale clustering of ErbB2. (A,B) SKBR-3 cells were labeled with FITC-CTX-B, Cy3-4D5 and Cy5-4D5 Fab. The FITC (green) and Cy3 (red) channels are overlaid in image A. Yellow areas correspond to colocalization between CTX-B-labeled lipid rafts and ErbB2 clusters. The homoassociation of ErbB2 was calculated using the acceptor photobleaching FRET method, and the FRET efficiency is displayed in panel B. FRET efficiency is color-coded according to the color-scale. The white boxes mark areas with inverse correlation between the local CTX-labeling density and ErbB2 homoassociation: the greener the area is in image A, the lower the FRET efficiency is in the corresponding part of the cell in image B. (C) Heregulin-stimulated SKBR-3 cells were labeled with FITC-CTX-B and Cy3-4D5 Fab. (D) SKBR-3 cells were treated with FITC-CTX-B at 37°C for 30 minutes, and they were subsequently labeled with Cy3-4D5 Fab. (E) SKBR-3 cells were pretreated with FITC-CTX-B and then incubated with Cy3-4D5 antibody at 37°C for 30 minutes. In part A, C and E the FITC image (green) is overlaid on the Cy3 (red) image. Yellow areas correspond to significant colocalization between lipid rafts and ErbB2 clusters. Bar, 1 µm.