Mig inhibits agonist-induced Rac activation and Rac2 is required for Mig's inhibitory activity. (A) Eosinophil lysates were used for affinity precipitation with 5 μg PAK-PBD for 60 minutes at 4°C. Active Rac-GTP precipitated by PAK-PBD was separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted for pan-Rac, followed by enhanced chemiluminescence (ECL) detection. In the bottom panel, aliquots of lysates were immunoblotted and probed for Rac to confirm equal protein expression. Representative blots are shown (n = 3). (B) Wild-type (WT, ♦) and Rac2-deficient (▪; KO indicates knock out) eosinophil transmigration toward eotaxin-2 is shown. Data represent mean ± SD of eosinophils that migrated toward eotaxin-2 (0-10 nM). A representative experiment is shown (n = 3). P = .03 between wild-type and Rac2^–/– at 1 and 10 nM based on paired Student t test. (C) Wild-type (□) and Rac2-deficient (▪) eosinophil transmigration toward eotaxin-2 is shown following pretreatment with buffer, eotaxin-2 (Etx2; 5 nM), or Mig (0.8-40 nM). Data represent mean ± SD of eosinophils that migrated toward eotaxin-2 (1 nM). A representative experiment is shown (n = 3). *P < .05 when compared with pretreatment of buffer alone. (D) Wild-type (solid lines) or Rac2-deficient (dashed lines) eosinophils were treated with 12 nM eotaxin-1 (♦), or 40 nM Mig and 12 nM eotaxin-1 (•) for the indicated period of time. Cells were fixed and stained with NBD-phallacidin. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone–stimulated cells.