Fig. 3. V12 Rac is adhesion dependent. Rat-1 cells stably expressing Myc-tagged V12 Rac were cultured for 48 h in the presence or absence of tetracycline (tet) and extracts prepared. (A) Lysates were analyzed by Western blotting with anti-Rac and anti-myc mAbs to assess cellular levels of the V12 and endogenous Rac. (B) Control Rat-1 cells or V12 Rac cells were cultured with or without tet in low serum, then placed in suspension for 2 h or kept stably adherent. PAK was then immunoprecipitated and its kinase activity determined using the in-gel kinase assay. Data are representative of three independent experiments. (C) Densitometric quantitation of PAK kinase activity normalized for PAK protein levels; values are means ± SD from three experiments.