Fig. 4. E-cadherin-mediated cell-cell adhesion inhibits Rap1 activity both in adherent and suspended cells. (A) Serum-starved FRT cell monolayers were subjected to the Ca²⁺ switch procedure either in the absence or presence of DECMA-1, a neutralizing antibody against E-cadherin, and further processed for measurement of active Rap1 by the pull-down assay. Notice that the decrease in Rap1 activity induced by restoration of cell-cell adhesion was inhibited by DECMA-1, indicating an E-cadherin function requirement. (B,C) Serum-starved FRT cells were harvested from tissue culture dishes and completely dissociated into single cells by 5 mM EDTA treatment and pipetting, and were then subjected to an aggregation assay (see Materials and Methods) either in the absence or in the presence of DECMA-1 to block E-cadherin function. Cells were then either analyzed by phase-contrast microscopy (B), or processed for measurement of active Rap1 (C). Notice the effectiveness of DECMA-1 in blocking both cell aggregation (B) and the E-cadherin function-dependent down-regulation of Rap1 activity (C) induced by Ca²⁺ restoration.