Figure 5.

Cdc42 and Rac1 activation exhibit a biphasic dependence on substrate concentration, whereas RhoA activation increases with increasing substratum concentration. (A) Representative blot showing that Cdc42 activation is optimal at an intermediate fibronectin concentration. Serum-starved CHO cells were kept in suspension (1 and 5) or plated onto low (2 and 6), intermediate (3 and 7), or very high (4 and 8) concentrations of fibronectin for 3 h and activity assays were performed as described in MATERIALS AND METHODS. Affinity-precipitated Cdc42-GTP was run in lanes 1–4 and total cell lysate was run in lanes 5–8. (B) Representative blot showing that Rac1 activation is optimal at an intermediate fibronectin concentration. Serum-starved CHO cells were plated onto low (1 and 4), intermediate (2 and 5), or very high (3 and 6) concentrations of fibronectin for 3 h and activity assays were performed as described in MATERIALS AND METHODS. Affinity-precipitated Rac1-GTP was run in lanes 1–3 and total cell lysate was run in lanes 4–6. (C) Representative blot showing that RhoA activation increases with increasing fibronectin concentration. Serum-starved CHO cells were kept in suspension (1 and 5) or plated onto low (2 and 6), intermediate (3 and 7), or very high (4 and 8) concentrations of fibronectin for 3 h and activity assays were performed as described in MATERIALS AND METHODS. Affinity-precipitated RhoA-GTP was run in lanes 1–4 and the total cell lysate was run in lanes 5–8.