Fig. 5. Membrane targeting is required for PAK activation. Normal V12 Rac or V12 Rac with a mutated CAAX sequence in the vector pBK-CMV were transiently transfected into NIH 3T3 cells. Transfection efficiency was ∼30%. (A) Western blots of lysate from transfected cells demonstrate equivalent expression of the Rac constructs. (B) At 24 h, cells were transferred to medium with 0.2% serum. At 48 h, the adherent cells were harvested and total endogenous PAK was immunoprecipitated. Both PAK protein and kinase activity were assayed and specific activity (kinase activity normalized for PAK protein) calculated. Values are means ± SD (n = 3).