Fig. 2. Regulation of Rho activity by cell adhesion to fibronectin. After starvation of cells in 0.5% serum for 20 hours, CHO (A), ß1 (B), ß3 (C) and ß1-3-1 (D) cells were trypsinized and kept in suspension in DMEM for 1 hour and then plated on culture dishes coated with fibronectin for various durations in the absence of serum. The amount of activated RhoA was determined by RBD pull down assay. The top panels in A-D show RBD-bound RhoA from cell lysates. The bottom immunoblot shows RhoA in whole cell lysates. For each cell type, the results of densitometric analysis of RBD-bound RhoA were normalized by the RhoA in whole cell lysates and plotted as a ratio of the results (mean±s.d. from three experiments). Asterisks indicate significant difference (P<0.05) from the ratio of 1. (E) RhoA activities of CHO, ß1, ß3 and ß1-3-1 cells in suspension, normalized for whole cell lysates and expressed as a ratio to CHO cells (mean±s.d. from three experiments). There was no significant difference among the four groups of cells in suspension.

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