Rap1b negatively regulates neutrophil transcellular migration by limiting PI3K-Akt signaling. (A–D) Effect of Akt inhibitor MK2206 (2 µM), Src inhibitor PP2 (10 µM), or vehicle control (DMSO) on WT or Rap1b^−/− neutrophil functions. (A) Percentage of neutrophil transendothelial migration in 3D migration model. (B) ECM degradation assessed on Oregon green–labeled gelatin matrix; (left) representative images on (bar, 10 μm) and (right) bar graph is percentage of matrix degradation. (C) Percentage of cells forming multiple protrusions. (D) Percentage of neutrophils present at junction of activated bEND.3 in 3D migration assay. Mean ± SD; n = 3 independent experiments. **, P < 0.01; ***, P < 0.001; NS, not significant using unpaired Student’s t test). (E and F) Effect of Akt inhibitor on neutrophil infiltration to inflamed lungs and survival of mice; Analysis of BAL at 4 h for neutrophil infiltration after interstitial LPS (1.25 mg/kg) challenge in mice (n = 4 mice per group from two independent experiments; mean ± SD; *, P < 0.05; **, P < 0.01). (E) Survival of mice reconstituted with WT or Rap1b^−/− BM (n = 6 per group, two independent experiments) injected i.p. with 20 mg/kg LPS (F), which were pretreated with MK2206 (15 mg/kg) or vehicle control DMSO I h before LPS challenge. ***, P = 0.00047 (Rap1b^−/− vs. MK2206 treatment, log-rank test).