Localization of cadherins in metastatic mammary carcinoma cells overexpressing wild-type and amino-terminal ezrin. Metastatic AC2M2 cells were transfected with empty pCB6 vector, or a vector encoding wild-type or amino-terminal ezrin, as described in the text. (a) Serial dilutions of the total cell extracts (0.6–20 μg) were subjected to reduced 10% SDS-PAGE and transferred to PVP membranes. The membranes were probed with anti-ezrin and anti-actin antibodies, followed by the appropriate peroxidase-conjugated secondary antibodies, and developed with chemiluminescence. Lanes from left to right contained 10 μg of the following cell extracts: pooled AC2M2 cells transfected with empty pCB6 vector, and two clones transfected with wild-type (WT) ezrin. WTC4 and WTC6 exhibited 4-fold and 8-fold overexpression, respectively, of ezrin compared with vector control cells. (b) Membranes were probed with anti-vesicular stomatitis virus glycoprotein (VSVG), anti-ezrin and anti-actin antibodies. Lanes contained 15 μg of cell extracts from pooled pCB6-transfected cells, and two clones transfected with amino-terminal ezrin. Clones NTC6 and NTC7 exhibited 1.6-fold and 4.6-fold amino-terminal ezrin expression, respectively, compared with endogenous ezrin, as determined by densitometric analysis of VSVG and ezrin blots normalized to actin. (c-g) The above cell lines were immunostained with anti-pan cadherin antibody, as described the text. Representative confocal microscope images are shown.