PIP3 distribution in chemotaxing neutrophils. (A) PIP3 immunolocalization to leading edge is disrupted in Rac1 null neutrophils. Immunolocalization of PIP3 was carried out and analyzed as described in “Materials and methods.” Representative images demonstrate the PIP3 localization toward the leading edge in wild-type and Rac2 null neutrophils but not in Rac1 null cells. (B) Immunoblot analysis of phospho-Akt levels in neutrophils. Representative immunoblot of Akt and phospho-Akt levels in peritoneal neutrophils. Neutrophils were isolated from bone marrow as described. Blot is representative of 4 separate experiments that all demonstrate decreased phospho-Akt levels in Rac1 and Rac1/2 null neutrophils compared to wild-type and Rac2 null neutrophils. (C) Immunolocalization of phospho-Akt to the leading edge requires Rac1. Immunolocalization of phospho-Akt, was carried out and analyzed as described in “Materials and methods.” Representative images demonstrate phospho-Akt localization toward the leading edge in wild-type and Rac2 null neutrophils but not in Rac1 null cells. *Leading edge. (D) Quantification of mean phospho-Akt–related fluorescence intensity at the leading edge, in the cytoplasm, and in the tail was carried out as described in “Materials and methods.” These data clearly confirm that p-AKT levels are high at the leading edge in both wild-type and Rac2 null neutrophils but decreased in Rac1 null cells. (The leading edge of Rac1 was different from both wild-type and Rac2 null at the P < .05 level.) Scale bar equals 5 μm. Error bars represent standard error of the means.
