Effect of Akt gene knockout on p47^phox phosphorylation and membrane translocation. (A) Neutrophils from WT, Akt1^−/−, and Akt2^−/− mice were stimulated for 1 minute with C5a (100nM). Total phosphoproteins were prepared by the use of antiphosphoprotein affinity chromatography as described in “Detection of p47^phox phosphorylation.” The p47^phox proteins in total lysate (total p47^phox) and in the phosphoprotein fraction (P-p47^phox) were detected by the use of an anti-p47^phox antibody. The relative intensity of the Western blot bands (phosphorylated p47^phox/total p47^phox) was quantified and shown in panel B; *P < .05; NS, no agonist stimulation. (C) Neutrophils from WT, Akt1^−/−, and Akt2^−/− mice were stimulated for 1 minute with C5a (100nM). Membrane fractions were prepared, separated and resolved on SDS-PAGE, and detected with specific antibodies recognizing p47^phox and p22^phox (a membrane protein used as membrane marker for loading control). Western blot bands (p47^phox/p22^phox) was quantified and shown in panel D. *P < .05; NS, no agonist stimulation.