Rap1b negatively regulates PI3K-Akt signaling. (A) Immunostaining analysis of PIP3 in WT and Rap1b^−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated surface. Bar, 10 µm. Fluorescence intensities were measured using Openlab software. Mean ± SD; n = 3 independent experiments. (B) Kinetics of Phospho-Akt (Ser473) in WT and Rap1b^−/− neutrophils using immunoblotting after fMLP stimulation and adhesion on fibrinogen-coated surfaces. Results of Akt phosphorylation expressed as the ratio of phospho-Akt to Akt as densitometry analysis. (C) Immunoblot analysis of p-Akt from WCL of neutrophils that were stimulated with fMLP in suspension. (D) Immunoblotting of p-p38 and p-Erk from WT and Rap1b^−/− neutrophils that were stimulated with fMLP and plated on fibrinogen-coated plates. (E) WT and Rap1b^−/− neutrophils were pretreated with indicated inhibitors for 20 min and stimulated with fMLP for 2 min. Akt activation was completely inhibited with Akt inhibitor MK2206 (2 µM) and, to a substantial level, with PI3K inhibitor Ly294002 (10 µM). Surprisingly, Src kinase inhibitor PP2 (10 µM) also inhibited Akt activation to a substantial level. Furthermore, rapamycin, an inhibitor of mTOR complex 1 failed to inhibit p-Akt. Although p-p38 and p-Erk were remained unchanged with these inhibitors, suggested specificity of p-Akt pathway. Blots are representative of three independent experiments (B–E). Densitometry analysis is cumulative of three experiments. Mean ± SD; *, P < 0.05; **, P < 0.01, using unpaired Student’s t test.