PTEN control of RAC activation.

(A) A recombinant PAK-CRIB domain–binding assay was used to determine levels of GTP-Rac 1 in cell lysates of COS7 cells transduced with FcγRIIA, Syk in the presence or absence of PTEN or mutant PTEN under conditions of FcγRIIA stimulation. The transfection condition for each group is shown above the lane. Lanes 1, 4, and 7 show no stimulation (NS); lanes 2, 5, 8, and 10, sRBC stimulation for 1 minute at 37° C; lanes 3, 6, and 9, stimulation of transfected COS7 cells for 3 minutes. Lane 10 shows vector control (VC), COS7 cells transfected with empty vectors pcDNA, pEGFP, and pRK5 and stimulated for 1 minute with sensitized sRBCs. Lane 11 shows positive control for GTP-Rac, a COS7 cell lysate incubated with GTPγS. (B) Anti-PTEN Western blot analysis of transfected COS7 cells analyzed for activation of Rac following FcγRIIA ligation with IgG-sensitized sRBCs. Lane 1 shows COS7 cells with no transfection; lane 2, transfection with all 3 empty vectors, pcDNA, pEGFP, and pRK5; lane 3, transfection with FcγRIIA, Syk, and pRK5; lane 4, transfection with FcγRIIA, Syk, and wildtype PTEN; and lane 5, transfection with FcγRIIA, Syk, and mutant PTEN (C124S).