Activation status of Gαi3 determines its role in cell migration. (A) Cell migratory behavior is restored by wild-type (a and b) and active (Q204L; c and d), but not the inactive (G203A; e and f), Gαi3 mutant. (Top) HeLa cells expressing rGαi3wt, active, or inactive mutants were subjected to scratch wounding. (Bottom) Equal overexpression of Gαi3 constructs was confirmed by immunoblotting. In assays in which siRNA was followed by plasmid overexpression, the efficiency of transfection (∼45–55%) was similar for wt, active, or inactive Gαi3 constructs by IF (n = 3). (B) Insulin-stimulated Akt activation is restored by wt and active (Q204L) but not the inactive (G203A) Gαi3 mutant. (Top) Assays were performed as in Fig. 4 (A and B) and samples were immunoblotted for tAkt and pAkt 5 min after stimulation. (Bottom) Bar graph showing percentage of Akt activation in cells treated as in top. Results are shown as mean ± SEM (n = 3). (C) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), mutant rGαi3-YFP restores normal actin organization (Phalloidin, red) in Gαi3-depleted cells. *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm. (D) Transfection of active (Q204L; a–c), but not inactive (G203A; d–f), rGαi3-YFP restores the normal (Fig. 4D, a) scattered peripheral and Golgi-associated (arrowheads) punctate distribution of GIV (red). *, cell expressing rGαi3-YFP visualized with anti-GFP (green). Bar, 10 μm.