Different cellular distribution and phosphorylation of Akt1 and Akt2. (A) WT mouse neutrophils were stimulated with 5μM fMLF (+) or buffer (−) for 2 minutes. The membrane and cytosolic fractions were separated, resolved on SDS-PAGE, and detected with specific antibodies recognizing Akt1 or Akt2. N.S. indicates a nonspecific species recognized by the anti-Akt1 antibody. Anti-p22^phox and anti–β-actin antibodies were used for detection of p22^phox and β-actin as markers of the membrane and cytosolic fractions, respectively. Tot indicates total (membrane plus cytosolic fractions); Cyt, cytosolic fraction; and Mem, membrane fraction. (B) Chemoattractant-induced Akt phosphorylation. WT, Akt1^−/−, and Akt2^−/− neutrophils (1 × 10⁷) from WT, Akt1^−/−, and Akt2^−/− mice were stimulated with C5a (+, 100nM) or buffer (−) for 1 minute. The samples were then resolved on SDS-PAGE and detected with specific anti–phospho-Akt antibodies recognizing phosphorylated Akt at Ser473 (Cell Signaling) and Thr308 (Calbiochem). β-actin and total Akt were detected for equal loading controls. Representative blots from 3 independent experiments are shown.