Akt phosphorylation and actin polymerization in PTEN^−/− neutrophils. (A-B) Suspensions of wild-type and PTEN^−/− neutrophils (0.8 × 10⁶ cells) were stimulated with 1 μM fMLP and lysed at 0, 60, 180, and 300 seconds. Protein lysates from 0.16 × 10⁶ cells were resolved on SDS–polyacrylamide gel electrophoresis (PAGE). Phosphorylated and total Akt were detected by Western blotting analysis using anti–Phospho-Akt (Ser473) (1:5000) and anti-Akt (1:1000) antibodies (Cell Signaling, Beverly, MA), respectively. (A) Results of the Western blots. (B) Results of densitometry. Akt phosphorylation expressed as ratio of phospho-Akt and total Akt. (C-D) Wild-type and PTEN^−/− neutrophils (0.5 × 10⁶ cells) were stimulated in suspension with 1 μM fMLP for 0, 60, 180, and 300 seconds; fixed; and stained with 0.13 μg/mL fluorescien phalloidin. Stained neutrophils were then analyzed by fluorescence-activated cell sorting (FACS). (C) Kinetics of F-actin polymerization. Phalloidin staining was represented as mean fluorescence at specified time normalized to mean fluorescence of unstimulated cells. The original FACS data were presented in Figure S3. (D) Decline in F-actin level was represented as the ratio of phalloidin fluorescence at 60 seconds (maximum) to fluorescence at 300 seconds. Data in panels C and D are represented as mean ± SD.