Fig. 7. pß-catenin is transiently associated with nascent adherens junctions. (a) MDCK cells were transfected with HA-ß-catenin and after 48 hours fixed and stained as described in the Materials and Methods. Ratio imaging analysis was carried out at six optical sections (0.6 µm apart) and shows exclusive localization of pß-catenin (red) in nuclear speckles and not in adherens junctions. (b) BCAP cells were plated on coverslips for the specified times (A-D) or treated with 4 mM EGTA for 1 hour (E) or washed and allowed to recover for 3 hours in the presence of fresh medium containing Ca²⁺ (F). The cells were fixed, stained and analyzed by ratio imaging analysis. The colors represent the ratio between pß-catenin (red) and either HA ß-catenin (blue, in the transfected MDCK cells) or total endogenous ß-catenin (blue, in the BCAP cells). The insert in A shows the specific inhibition of the pß-catenin staining in the presence of the double phosphorylated peptide (pep). Bar, 10 µm.