IL-3 activates multiple Rho family GTPases in BMMCs.

BMMCs generated from BDF1 mice were incubated for 5 minutes with PBS (con) or IL-3 (5 μg/mL). Cells were lysed and GST-PAK was used to precipitate endogenous, GTP-bound Rac-1, Rac-2 or Cdc42. Immunoblots were performed with (A) a Rac-1 specific monoclonal antibody, (B) polyclonal antibodies specific for Rac-2, or (C) polyclonal antibodies specific for Cdc42. Immunoblots of Rac/Cdc42 present in WCLs were performed with the same antibodies as used to visualize Rac/Cdc42-GTP precipitated with GST-PAK (except for the case of Rac-2 where, for technical reasons, the Rac-1 antibody was used to probe the immunoblot of the WCL). One half of total GTP-bound Rac-1 precipitated was run in the Rac-1 immunoblot; the WCL corresponded to 3% of total Rac-1 available for precipitation. The total amount of GTP-bound Cdc42 precipitated was run in the Cdc42-specific immunoblot; the WCL corresponded to 4% of the total Cdc42 available for precipitation. Shown below immunoblots GTP-bound GTPases in this and succeeding figures are numbers corresponding to the ratios of the optical densities of the bands from stimulated versus control cells.