Fig. 8. Expression and localization of N-cadherin and associated proteins among immortalized fibroblasts derived from wild-type and fer^D743R mice. Fibroblasts derived from wild-type (WT) or fer^D743R (D743R) embryos were cultured to confluence, washed free of serum and lysed in mild detergent buffer. (A) Equal amounts of protein from cell lysates were analysed by western blot using the indicated antibodies. (B) Equal amounts of protein were immunoprecipitated with anti-pan-cadherin, anti-ß-catenin or anti-PTP1B antibodies and the immunoprecipitates analysed by western blot using the indicated antibodies. (C) WT or D743R cells were stained with Alexa-568/phalloidin and anti-pan-cadherin or anti-ß-catenin antibodies, as indicated, followed by the appropriate Alexa-488-conjugated second antibody, and visualized by confocal microscopy. Among WT cells, cadherin `zippers' are clearly seen at cell boundaries, where actin and cadherin or actin and ß-catenin overlap (arrowhead). In D743R cells, there is no detectable cadherin or ß-catenin at cell boundaries (arrowhead). Scale bar, 10 µm. (D) Aggregation of WT and D743R cells in suspension cultures. Single cells from WT or D743R cultures were prepared by trypsin dissociation and allowed to aggregate in the presence of 1 mM Ca²⁺ or 5 mM EGTA for 3 hours at 37°C and with shaking at 80 rpm, in 30 mm dishes. Cells were visualized under phase-contrast and photographed using an Axiovert25 microscope and Axiovision system (Zeiss).