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Fig. 1. Rap1 activity is gradually down-regulated during cell adhesion independently of integrin activation and function. (A) Confluent cells kept overnight in serum-free medium were either lysed directly onto tissue culture dishes (Ad, adherent cells), or harvested by treatment with 5 mM EDTA in PBS and resuspended in serum-free medium before lysis (Su, suspension cells). Active, GTP-bound Rap1 (GTP-Rap1) was detected by a pull-down assay using a GST-tagged RalGDS-RBD protein prebound to glutathione-Sepharose beads (see Materials and Methods); western blotting of whole lysates ensured that relative equal amounts of total Rap1 proteins (Total Rap1) were analyzed. Notice that cell detachment from the substratum promoted a strong activation of Rap1 in all cell lines tested. (B-D) Resuspended cells were either lysed (Su, suspension cells), or replated at high density and allowed to adhere for the indicated times (in minutes) to tissue culture dishes coated with 10 µg/ml of the substratum proteins, fibronectin (FN) (B), poly-L-lysine (PL) (C) or an activating mAb to ß₁ integrin (TS2/16) (D), before measurement of active Rap1. Notice that cell adhesion induced a progressive down-regulation of Rap1 activity, which was independent of the adhesive substratum used and reached the maximal level at about 1 hour.

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