Specificity of Rac antibodies and activation of Rac-1 by β-common family cytokines.

Baf/3 cells were electroporated with 15 μg constitutively active Rac-1 (Rac-1 myc) or Rac-2 (Rac-2 myc). At 16 hours after electroporation, cells were lysed and both overexpressed and endogenous Rac GTPases were precipitated using recombinant GST-PAK bound to GT beads. Antibodies specific for Rac-1 or Rac-2 were used for immunoblotting serial 2-fold dilutions (1, ½, ¼) of lysates with (A) overexpressed, myc-tagged Rac-1 or Rac-2 as indicated or (B) endogenous and overexpressed Rac-1 or Rac-2 isoforms. (C) MC/9 cells were incubated for 5 minutes with PBS (con), GM-CSF (10 μg/mL), IL-3 (5 μg/mL), or IL-5 (50 ng/mL) and lysed. Immunoblots of Rac-1 GTP bound by beads and in WCLs were performed using a Rac-1–specific monoclonal antibody (Rac-1 GTP). One half of the total GTP-bound Rac-1 precipitated was run in immunoblots. The WCLs corresponded to 1.3%, 1.2%, and 1.2% of the total Rac available for precipitation for GM-CSF, IL-3, and IL-5, respectively. Numbers below immunoblots of Rac-1 GTP (and of other GTP-bound GTPases in succeeding figures) represent the ratios of the optical density of the immunoblotted band of GST-PAK precipitated Rac-1 from stimulated cells to the optical density of the immunoblotted band of GST-PAK precipitated Rac-1 from control, unstimulated cells.