FIG. 7.

PKCδ activity regulates GTP-Rac translocation to the membrane. (A) COS-7 cells were transfected as indicated with PLD2 siRNA or with luciferase siRNA as a control. After 24 h of siRNA transfection, cells were cotransfected with Flag-V12-Rac1 and either an empty vector, PLD2^WT, PLD2^T566A, or PLD2^T566E, as indicated. After serum starvation for 24 h, cells were detached from culture dishes, maintained in suspension for 3 h, and replated on PLL or FN for 30 min. Adherent cells were harvested, and particulate membrane fractions were isolated. Samples were analyzed by Western blotting using an anti-pan-PLD or anti-Flag antibody. β1 integrin was used as a membrane fraction marker. The amounts of Rac1 in the membrane determined by densitometry were normalized to the amount of β1 integrin in the membrane. The results shown are representative of three independent experiments. (B) COS-7 cells treated as described for panel A were harvested, and Rac GTP loading was quantified by PBD pulldown assays as described in Materials and Methods.