Figure 4.

Imatinib does not affect GTP-Rac or GTP-Rho levels during cell spreading. (A) 3T3 fibroblasts were serum starved for 18 h before detachment. Cells were held in serum free DMEM for 30 min and then treated with imatinib (5 μM) or vehicle (0.1% DMSO) for 45 min. Cells were plated on FN-coated dishes for the indicated time and harvested for Rac pull-down with GST-PBD as described in Materials and Methods. (B) Quantitation of data in A as described in Materials and Methods. The ratio of GST-PBD pull-downed Rac over total Rac of the vehicle-treated 5-min sample was set to 1. Values shown are from three independent experiments. (C) Cells were processed and harvested as in A and Rho was pulled down with GST-RBD (Rho-binding domain of Rhotekin) as described in Materials and Methods. (D) Quantitation of GST-RBD pull-down Rho as in B. Values are from three independent experiments.