Figure 6

Confocal microscopy images of internalization of rr1-cadherin antibody-labeled cell surface cadherin in v-Src transformed MDCK cells grown at +40.5°C in cell culture dishes to confluency, surface-labeled on ice with rr1 antibody and warmed for 30 min or 60 min to +40.5°C or +35°C before fixation. Cadherin is shown in green, β-catenin in red and nuclei blue. In cells fixed on ice, rr1 staining was seen at lateral membranes near apical surface. In 30 min after warming to +40.5°C or +35°C, rr1-labelled E-cadherin was seen in the cell interior in apical vesicles void of β-catenin (arrow head), but co-localizing with β-catenin at basal surfaces. Within 60 min after warming to +40.5°C, rr1-labelled E-cadherin partially accumulated back to lateral membranes, partially remained in apical vesicles (arrow head), whereas in cells incubated at 35°C, much less rr1-conjugated cadherin was found at lateral membranes and the majority remained in cytoplasm and in apical vesicles void of β-catenin (arrow head). ROIs occasionally seen in images were used for the quantitative co-localization analysis presented in Table 1. Bar: 20 μm.